PMMA binding peptides and methods of use

ABSTRACT

Combinatorially generated peptides are provided that have binding affinity for polymethylmethacrylate (PMMA). The peptides may be used to deliver benefit agents to various PMMA surfaces.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. §119 from U.S.Provisional Application Ser. No. 60/750,548, filed Dec. 15, 2005.

FIELD OF THE INVENTION

The invention relates to peptide based reagents having binding affinityfor polymethylmethacrylate polymers.

BACKGROUND OF THE INVENTION

Polymethylmethacrylate (PMMA) is a clear polymer developed as a glasssubstitute. It is commonly referred to as acrylic glass or acrylic andmarketed under trademarks such as: Plexiglas™, Perspex™, Acrylite™,Acrylplast™ and Lucite™. PMMA has several advantages over silicon glass.Its density is less than half of its silicon counterpart. PMMA does notshatter. It can be formed at relatively low temperatures. These andother qualities have led engineers to adapt PMMA to other purposesbeyond glass replacement. Nevertheless, PMMA is still commonly used forits original purpose, it is found in large windows, aquariums, andvehicle rear lights. However, it is also found in places that glasswould never be considered a suitable material. For example it is used asa bone replacement, dentures, and paint coating.

Some physical properties of PMMA are undesirable for a givenapplication. In theses circumstances a coating is applied to mask theundesirable property. For example, PMMA is hydrophobic, when used as anintraocular lens it must be coated to make the surface hydrophilic.Also, PMMA tends to carry a static charge, in some circumstances thecharge can build to the point of fracture. DuPont's Zelek-NK™ antistaticcoating was developed to counter this tendency.

The ubiquitous use of PMMA in industry makes it a prime materialcandidate for a variety of applications where the PMMA comprises some orall of a surface. One of the drawbacks to using PMMA as surface is thatmaterials that bind to PMMA are specific and lack flexibility as bindingagents. So for example where a new coating for PMMA is desired, a newsearch for a PMMA binding molecule with the desired property must beconducted. The resulting search is costly in both time and resources andnot guaranteed to be successful. A system that is flexible and can beeasily tailored for a variety of materials to be bound to PMMA isneeded. The use of peptides as linkers or binders to PMMA offers somepotential in this regard.

Peptides having a binding affinity to polymer and plastic surfaces areknown. For example, Adey et al., (Gene 156:27-31 (1995)) describepeptides that bind to polystyrene and polyvinyl chloride surfaces.Additionally, peptides that bind to polyurethane (Murray et al., U.S.Patent Application Publication No. 2002/0098524), polyethyleneterephthalate (O'Brien et al., copending and commonly owned U.S. PatentApplication Publication No. 2005/0054752), and polystyrene,polyurethane, polycarbonate, and nylon (Grinstaff et al., U.S. PatentApplication Publication No. 2003/0185870) have been reported. However,the use of such peptides to target PMMA surfaces has not been described.

There remains a need therefore for a peptide based reagent that bindsPMMA that offers flexibility in bring a wide variety of materials to thePMMA surface with minimum investment in redesign. Applicants have solvedthe stated problem by providing peptide reagents comprising PMMA bindingpeptides (PmBP). The PMMA binding peptides of the invention may bemodified with other functional or binding peptides allowing for thedelivery of benefit agents to the PMMA surface or for the use of thereagents to adhere PMMA containing surfaces.

SUMMARY OF THE INVENTION

The present invention provides PMMA binding peptides that may beincorporated into peptide based reagents useful for deliveringfunctional compounds to a PMMA surface. The PMMA binding peptides maycomprise active domains that have linker or other functionality ortarget binding domains that bind various benefit agents that aredelivered to the PMMA surface.

Accordingly, in one embodiment the invention provides a peptide reagenthaving a general structure selected from the group consisting of:PMMA_(m)-(PmBP)_(n);  a)PMMA_(m)-(PmBP-BA_(p))_(n);  b)PMMA_(m)-(PmBP-AD)_(n);  c)PMMA_(m)-(PmBP-TBD)_(n); and  d)PMMA_(m)-(PmBP-L-BA)_(n); and  e)PMMA_(m)-[(PmBP)_(q)-(L)_(x)-(PmBP)_(r)]_(n)-L-BA;  f)

wherein:

-   -   i) PMMA is a PMMA moiety    -   ii) PmBP is a PMMA binding peptide having a PMMA binding domain;    -   iii) BA is at least one benefit agent;    -   iv) AD is at least one active domain incorporated into a PMMA        binding peptide;    -   v) TBD is at least one target binding domain incorporated into a        PMMA binding peptide;    -   vi) L is a linker molecule;    -   vii) m=the number of PMMA moieties available for binding;    -   viii) n=is less than or equal to m;    -   xi) p=1-20;    -   x) x=1-20; and    -   xi) r=1-50.    -   In an alternate embodiment the invention provides a peptide        reagent having the general structure:        (BA)_(n)-(L)_(m)-PMMA_(p)-[(X)_(a)-(Y)_(b)]_(q)-(L)_(r)-(BA)_(s)    -   Wherein:    -   i) BA is a benefit agent;    -   ii) PMMA is a PMMA moiety;    -   iii) L is a linker molecule;    -   iv) X is a PMMA peptide binding domain;    -   v) Y is an active domain; and    -   vi) wherein a, b, m, n, p, q, r, and s are non-negative integers        wherein, b, n, r, and s may be 0; and        -   a, p and q will at least be 1.

In another embodiment the invention provides a method for binding asubstrate comprising PMMA to a target comprising:

-   -   a) providing a peptide reagent of the invention: and    -   b) contacting the peptide reagent of (a) with a substrate        comprising a PMMA moiety under conditions whereby the peptide        reagent binds to the PMMA moiety.

Similarly the invention provides a method for delivering a benefit agentto a substrate comprising PMMA comprising:

-   -   a) providing the peptide reagent of the invention having a        benefit agent: and    -   b) contacting the peptide reagent of (a) with a substrate        comprising a PMMA moiety under conditions whereby the peptide        reagent binds to the PMMA moiety, whereby the benefit agent is        delivered to the substrate.    -   In an alternate embodiment the invention provides a method for        adhering two surfaces comprising:    -   a) providing a first surface comprising PMMA comprising a first        peptide regent having the general formula;        (PmBP-AD1)    -   wherein:        -   i) PmPB is a PMMA binding peptide; and        -   ii) AD1 is a first active domain;    -   b) providing a second surface comprising a target molecule        comprising a second peptide reagent have the general formula;        (TBP-AD2)        -   wherein:            -   iii) TBP is a target binding peptide; and            -   iv) AD2 is a second active domain having affinity for                the first active domain;    -   c) juxtaposing the first and second surfaces wherein the first        and second peptide reagents adhere to each other through the        first and second active domains, whereby the surfaces are        adhered.    -   In a similar embodiment the invention provides a method for        adhering two surfaces comprising:    -   a) providing a first surface comprising a first target molecule        comprising a first peptide regent having the general formula;        (TBP1-AD)        -   wherein:            -   i) TBP1 is a first target binding peptide; and            -   ii) AD is an active domain having binding affinity for                PMMA;    -   b) providing a second surface comprising a second target        molecule comprising a second peptide reagent have the general        formula;        (TBP2-AD)        -   wherein:            -   iii) TBP2 is a second target binding peptide; and            -   iv) AD is an active domain having binding affinity for                PMMA;    -   c) juxtaposing the first and second surfaces in the presence of        a PMMA moiety wherein the first and second peptide reagents        adhere to the PMMA moiety through the active domain, whereby the        surfaces are adhered.

Additionally the invention provides a PMMA binding peptide having anamino acid sequence selected from the group consisting of SEQ ID NO's:1-12.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS

FIG. 1 is a set of panels A-E which depict embodiments of the presentinvention as they are bound to a surface containing, in whole or inpart, PMMA particles.

FIG. 2 is a set of panels A-C which depict embodiments of the presentinvention as they are bound to a PMMA coating containing, in whole or inpart, PMMA particles, which is further bound to a surface.

FIG. 3 depicts an embodiment of the present invention as a diblock,optionally bound to a benefit agent at two different positions and/or atarget molecule. Also depicted is the optional inclusion of a linkermolecule and/or an active domain.

FIG. 4 depicts an embodiment of the present invention used to bond aPMMA containing surface with another surface which may contain PMMA oranother known target molecule.

FIG. 5 depicts an embodiment of the present invention used to bond tosurfaces together wherein neither necessarily contains PMMA.

FIG. 6 is a set of panels A-D which depict embodiments of the presentinvention used to coat a surface with PMMA.

SEQ ID NOs: 1-12 are PMMA binding peptide sequences.

SEQ ID NOs:13-41 are antimicrobial peptides sequences.

SEQ ID NOs: 42-66 are pigment binding peptides sequences.

SEQ ID NOs: 67-79 are print media binding peptide sequences: SEQ ID NOs:67 and 68 bind to cotton fabric, SEQ ID NOs: 67 and 69 bind topolyester/cotton fabric, SEQ ID NOs: 67, and 70-72 bind to HAMERMILL®paper, SEQ ID NOs: 74-78 bind to cellulose, and SEQ ID NO: 79 binds topoly(ethylene terephthalate).

SEQ ID NOs: 80-175 are body surface binding peptide sequences: SEQ IDNOs: 80-87 are skin-binding peptide sequences, SEQ ID NOs: 88-175 arehair binding peptide sequences and SEQ ID NOs: 88 and 89 bind nails aswell as hair.

SEQ ID NO:176 is the amino acid sequence of the Caspase 3 cleavage sitethat my be used as a peptide linker domain.

SEQ ID NOs: 177-179 are amino acid sequences of peptide linker domains.

A Sequence Listing is provided herewith on Compact Disk. The contents ofthe Compact Disk containing the Sequence Listing are hereby incorporatedby reference in compliance with 37 CFR 1.52(e). The Compact Disks aresubmitted in triplicate and are identical to one another. The disks arelabeled “Copy 1—Sequence Listing”, “Copy 2—Sequence Listing”, and CRF.The disks contain the following file: CL2828.ST25 having the followingsize: 40,000 bytes and which was created Nov. 20, 2006.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides variable coatings for PMMA substrates andsurfaces. More specifically, the present invention provides peptidesequences that bind PMMA with a high affinity. These peptides can bebound covalently or otherwise to known substances to adapt PMMA for avariety of uses. Additionally, the present invention provides methods todevelop and produce such peptides.

The following definitions and abbreviations are to be used for theinterpretation of the claims and the specification.

“BA” means benefit agent.

“PMMA” means polymethylmethacrylate

“PmBP” is a PMMA binding peptide

“PBP” means pigment-binding peptide.

The term “peptide” refers to two or more amino acids joined to eachother by peptide bonds or modified peptide bonds.

The term “body surface” will mean any surface of the human body that mayserve as a substrate for the binding of a peptide carrying a benefitagent. Typical body surfaces include but are not limited to hair, skin,nails, teeth, gums, and corneal tissue.

The term “benefit agent’ is a general term applying to a compound orsubstance that may be coupled with a complex of PMMA and PMMA bindingpeptide in order to provide a desirable characteristic of the benefitagent to the complex. In the most general sense a benefit agent may beany element, molecule or compound that is not PMMA or a PMMA-bindingpeptide. Benefit agents typically include colorants such as pigments anddyes as well as pharmaceuticals, markers, conditioners, and fragrances.

The term “hair” as used herein refers to human hair, eyebrows, andeyelashes.

The term “skin” as used herein refers to human skin, or pig skin,Vitro-Skin® and EpiDerm™ which are substitutes for human skin. Skin asused herein as a body surface will generally comprise a layer ofepithelial cells and may additionally comprise a layer of endothelialcells.

The term “nails” as used herein refers to human fingernails andtoenails.

The terms “coupling” and “coupled” as used herein refer to any chemicalassociation and includes both covalent and non-covalent interactions.

The term “pigment” refers to an insoluble, organic or inorganiccolorant.

The term “print medium” refers to any substrate suitable for printing.

The term “dispersant” as used herein refers to a substance thatstabilizes the formation of a colloidal solution of solid pigmentparticles in a liquid medium.

The term “PMMA binding peptide” refers to a peptide having specificaffinity for PMMA. The PMMA binding peptide will typically be shortranging from about 7 to about 50 amino acids in length and may begenerated recombinantly, synthetically or may be selected bycombinatorial means. PMMA binding peptides may comprise varioussubdomains including but not limited to active domains, target domainsand linker domains. Within any given PMMA binding peptide there residesa “PMMA binding domain” having affinity to PMMA. Any given PMMA bindingpeptide may contain only the PMMA binding domain or may contain thisdomain in conjunction with active or target domains having differentfunctionality.

The term “active domain” as used herein applies to a subsequence ofamino acids within a PMMA binding peptide. An active domain is a portionof the PMMA binding peptide that is not responsible for PMMA binding butprovides additional functionality or benefit. In one embodiment forexample an active domain may have antimicrobial functionality. In antherembodiment the active domain may have a linker function between twoother domains or between the peptide and a benefit agent. In anotherembodiment the active domain may serve to bind a specific target analyte(target domain).

The term “linking domain” or “linker domain” as used herein applies to aparticular of active domain that is used to either link two domainstogether, as a separator between two domains, or a domain and a terminalend. Linking domains may have a function beyond joining or separatingtwo features of a peptide.

The term “target binding domain” as used herein applies to a particulartype of peptide active domain that binds a target molecule, element,compound, or complex. The binding substrate for the target bindingdomain is referred to herein as the “target”. Typical targets willinclude but are not limited to biological analytes, (cells, cellmembrane fractions, viral proteins, proteins, antibodies, antibodyfragments, nucleic acids and the like), plant fibers, synthetic fibers,as well as organic and inorganic target complexes that will typically befound on surfaces or in print media. All target binding domains areactive domains. A “body surface binding domain” is a target domain thathas specific affinity for a body surface such as hair, skin, nails,teeth and the like. Similarly a “print media binding domain” willfunction to bind the elements of print media such as paper and other inkreceptive surfaces. Within the context of print media domains there maybe those domains that bind cellulose or cotton or other plant fibers.Additionally the target domains of the invention may be selected to bindspecific benefit agents such as colorants (pigments, dyes) andconditioners or any other organic or inorganic complex.

“PMMA moiety” means a discrete substance comprisingpolymethylmethacrylate that serves as a binding site for a PMMA bindingpeptide. PMMA moieties may make up a PMMA film, or be comprised withinvarious PMMA coatings or surfaces and substrates.

The term “linker” or “spacer” or ‘linker molecule” or “spacer molecule”will be used interchangeably and will mean a molecule or compound usedto bind a benefit agent to the PMMA-peptide complex. Any material thatcan bind said benefit agent to the complex can be used, includingpeptide based molecules. A linker molecule is distinct from a linkerdomain in that linker domains are inherently part of, or are proposed tobe part of a peptide further comprising a PMMA-binding domain. A linkermolecule, in whole or in part, may be identical to a linking domain, buta linking molecule does not contain a PMMA-binding domain.

As referred to herein a substance has “binding functionality” when itdemonstrates specific affinity for a substance or target.

As referred to herein a substance has “catalytic functionality” when itdemonstrates the ability to catalyze a chemical reaction

As referred to herein a substance has “antimicrobial functionality whenit demonstrates the ability to kill microbial cell populations.

As used herein the term “surface” when used in conjunction with a PMMAmoiety means the point of contact for the PMMA moiety. Surfaces of theinvention will typically be coated with PMMA or may themselves comprisePMMA moieties. Surfaces may take the form of solid support, a bead, amicrosphere, a sheet, or a fiber. In some instances the surface of theinvention may be layered or juxtaposed on a “secondary surface”. A“secondary surface” will typically be coated or layers with the PMMAsurfaces of the invention.

The term “diblock structure” as used herein refers to a composition thatconsists of two different units or blocks, each serving a specificfunction. The peptide-based diblock polymers of the present inventionconsist of a PMMA-binding peptide block coupled to a substrate, or aPMMA-binding peptide block coupled to a benefit agent. The diblockpolymer may contain multiple copies of the peptide block.

The term “triblock structure” as used herein refers to a pigmentdispersant that consists of three different units or blocks, eachserving a specific function. The peptide-based triblock structure of thepresent invention consists of a substrate-block, PMMA-binding peptideblock, and a benefit agent block. The triblock structure may containmultiple copies of any of the peptide blocks.

The term “stringency” as it is applied to the selection of PMMA bindingpeptides, hair-binding, skin-binding, and nail-binding peptides of thepresent invention, refers to the concentration of the eluting agent(usually detergent) used to elute peptides from the substrate to whichthey are bound or for which they have affinity. Higher concentrations ofthe eluting agent provide more stringent conditions.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The following abbreviations are used herein toidentify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments which are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing site, effector binding site andstem-loop structure.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The term “host cell” refers to cell which has been transformed ortransfected, or is capable of transformation or transfection by anexogenous polynucleotide sequence.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

The term “phage” or “bacteriophage” refers to a virus that infectsbacteria. Altered forms may be used for the purpose of the presentinvention. The preferred bacteriophage is derived from the “wild” phage,called M13. The M13 system can grow inside a bacterium, so that it doesnot destroy the cell it infects but causes it to make new phagescontinuously. It is a single-stranded DNA phage.

The term “phage display” refers to the display of functional foreignpeptides or small proteins on the surface of bacteriophage or phagemidparticles. Genetically engineered phage may be used to present peptidesas segments of their native surface proteins. Peptide libraries may beproduced by populations of phage with different gene sequences.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L.and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Cold Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987).

The present invention relates to peptides and peptide reagents that havespecific binding affinity to PMMA in various conformations includingcomplexes of the PMMA binding peptides linked to benefit agents, andoptionally where the PMMA binding peptides comprise active peptidedomains or target binding domains having binding or other functionalityfor other substances or surfaces. The PMMA peptide regent of theinvention may take a variety of forms including those represented by thefollowing structures:PMMA_(m)-(PmBP)_(n);  a)PMMA_(m)-(PmBP-BA_(p))_(n);  b)PMMA_(m)-(PmBP-AD)_(n);  c)PMMA_(m)-(PmBP-TBD)_(n); and  d)PMMA_(m)-(PmBP-L-BA)_(n); and  e)PMMA_(m)-[(PmBP)_(q)-(L)_(x)-(PmBP)_(r)]_(n)-L-BA;  f)

wherein:

-   -   i) PMMA is a PMMA moiety    -   ii) PmBP is a PMMA binding peptide having a PMMA binding domain;    -   iii) BA is at least one benefit agent;    -   iv) AD is at least one active domain incorporated into a PMMA        binding peptide;    -   v) TBD is at least one target binding domain incorporated into a        PMMA binding peptide;    -   vi) L is a linker molecule;    -   vii) m=the number of PMMA moieties available for binding;    -   viii) n=is less than or equal to m;    -   xi) p=1-20;    -   x) x=1-20; and    -   xi) r=1-50.

Alternatively the PMMA peptides reagents of the invention may beconfigured according formula:(BA)_(n)-(L)_(m)-PMMA_(p)-[(X)_(a)-(Y)_(b)]_(q)-(L)_(r)-(BA)_(s)

-   -   Wherein:    -   i) BA is a benefit agent;    -   ii) PMMA is a PMMA moiety;    -   iii) L is a linker molecule;    -   iv) X is a PMMA peptide binding domain;    -   v) Y is an active domain; and    -   vi) wherein a, b, m, n, p, q, r, and s are non-negative integers        wherein, b, n, r, and s may be 0; and        -   a, p and q will at least be 1.            PMMA Moieties

A PMMA (poly(methyl methacrylate)) moiety, as defined herein, is thebinding site of a PMMA-binding peptide on a surface. The PMMA moiety maybe incorporated into a surface in various ways. For example, the surfacemay be the surface of a PMMA substrate. Alternatively, the PMMA moietymay be imbedded into the surface of another material, such as a polymer,or coated on the surface of another material, such as a metal, polymer,glass, cloth, and the like. The PMMA moiety may comprise a PMMA polymeror a copolymer of methyl methacrylate with one or more monomers such asother acrylates, styrene, acrylonitrile, vinyl acetate, vinyl chloride,vinylidene chloride, and butadiene.

PMMA is prepared by the polymerization of the monomer methylmethacrylate, which is available from many commercial suppliers, such asAldrich (Milwaukee, Wis.), ICI Acrylics (Beaumont, Tex.), CYROIndustries (Rockaway, N.J.), Total Specialty Chemicals, Inc (New Canaan,Conn.), and Degussa Corp. (Parsippani, N.J.). Methyl methacrylate may bepolymerized using methods known in the art, such as radicalpolymerization, anionic polymerization, or group transfer polymerization(Ullmann's Encyclopedia of Industrial Chemistry, 6^(th) edition, 2003,Wiley-VCH Verlag GmbH and Co., Weinheim, Germany, Vol. 28, pp. 377-389).For example, radical polymerization may be carried out homogeneously(i.e., bulk or solution polymerization) or heterogeneously (i.e.,suspension or emulsion polymerization). The radical polymerization maybe initiated using radiation, heat, or chemical initiators, such as azocompounds or organic peroxy compounds. Copolymers may be produced bythese methods using a mixture of the desired monomers.

The PMMA polymer may be produced in various shapes or forms, such asbeads, microspheres, sheets, rods, tubes, films, plates, rings, fiber,and microfilament, using injection molding, extrusion, and castingtechniques, which are well known in the art. Additionally, PMMA invarious shapes is available commercially from companies such as CRYOIndustries and Bang Laboratories (Fishers, Ind.).

In one embodiment, the PMMA polymer or copolymer is coated onto anothersurface, such as metal, polymer, glass, cloth, and the like, usingmethods known in the art, such as spraying, brushing, dip coating andcasting.

In another embodiment, the PMMA polymer or copolymer is imbedded intothe surface of another material, such as another polymer. This may bedone by adding particles, beads, or fragments of PMMA material into theother polymer as it cures.

In another embodiment, a PMMA copolymer is used as a dispersant forpigments or other insoluble particles, including metallic andsemiconductor nanoparticles. The copolymer may be a random copolymer ora structured copolymer (i.e., a nonrandom block copolymer). Preferredrandom dispersants include methyl methacrylate copolymers with otheracrylates or styrene. Most preferred are structured polymer dispersants,which include AB, BAB and ABC block copolymers, branched polymers and,graft polymers. Preferably these copolymers comprise methyl methacrylatewith one or more monomers such as acrylate, methacrylate, butylmethacrylate, 2-ethylhexyl methacrylate, benzyl methacrylate,phenoxyethyl acrylate, and ethoxytriethyleneglycolmethacrylate, such asthose described by Nigan (U.S. Patent Application Publication No.2004/0232377). Some useful structured polymer dispersants are disclosedin U.S. Pat. No. 5,085,698, EP-A-0556649 and U.S. Pat. No. 5,231,131.

Identification of PMMA-Binding Peptides

Peptides having affinity for PMMA, referred to herein as PMMA-bindingpeptides (PmBP), are peptide sequences that bind strongly to a PMMAmoiety. The PMMA-binding peptides of the invention are from about 7amino acids to about 50 amino acids, more preferably, from about 7 aminoacids to about 25 amino acids, most preferably from about 7 to about 20amino acids in length. Suitable PMMA-binding peptides may be selectedusing methods that are well known in the art.

The PMMA-binding peptides may be generated randomly and then selectedagainst a PMMA substrate based upon their binding affinity for PMMA, asdescribed by O'Brien et al. (copending and commonly owned U.S. PatentApplication Publication No. 2005/0054752), Adey et al., (Gene 156:27-31,(1995)), Murray et al. (U.S. Patent Application Publication No.2002/0098524) and Grinstaff et al. (U.S. Patent Application PublicationNo. 2003/0185870), all of which are incorporated herein by reference.The generation of random libraries of peptides is well known and may beaccomplished by a variety of techniques including, bacterial display(Kemp, D. J.; Proc. Natl. Acad. Sci. USA 78(7):4520-4524 (1981), andHelfman et al., Proc. Natl. Acad. Sci. USA 80(1):31-35, (1983)), yeastdisplay (Chien et al., Proc Natl Acad Sci USA 88(21):9578-82 (1991)),combinatorial solid phase peptide synthesis (U.S. Pat. No. 5,449,754,U.S. Pat. No. 5,480,971, U.S. Pat. No. 5,585,275, U.S. Pat. No.5,639,603), and phage display technology (U.S. Pat. No. 5,223,409, U.S.Pat. No. 5,403,484, U.S. Pat. No. 5,571,698, U.S. Pat. No. 5,837,500).Techniques to generate such biological peptide libraries are well knownin the art. Exemplary methods are described in Dani, M., J. of Receptor& Signal Transduction Res., 21(4):447-468 (2001), Sidhu et al., Methodsin Enzymology 328:333-363 (2000), and Phage Display of Peptides andProteins, A Laboratory Manual, Brian K. Kay, Jill Winter, and JohnMcCafferty, eds.; Academic Press, NY, 1996. Additionally, phage displaylibraries are available commercially from companies such as New EnglandBioLabs (Beverly, Mass.).

A preferred method to randomly generate peptides is by phage display.Phage display is an in vitro selection technique in which a peptide orprotein is genetically fused to a coat protein of a bacteriophage,resulting in display of fused peptide on the exterior of the phagevirion, while the DNA encoding the fusion resides within the virion.This physical linkage between the displayed peptide and the DNA encodingit allows screening of vast numbers of variants of peptides, each linkedto a corresponding DNA sequence, by a simple in vitro selectionprocedure called “biopanning”. In its simplest form, biopanning iscarried out by incubating the pool of phage-displayed variants with atarget of interest that has been immobilized on a plate or bead, washingaway unbound phage, and eluting specifically bound phage by disruptingthe binding interactions between the phage and the target. The elutedphage is then amplified in vivo and the process is repeated, resultingin a stepwise enrichment of the phage pool in favor of the tightestbinding sequences. After 3 or more rounds of selection/amplification,individual clones are characterized by DNA sequencing.

Specifically, the PMMA-binding peptides may be selected using thefollowing method. A suitable library of phage-peptides is generatedusing the methods described above or the library is purchased from acommercial supplier. After the library of phage-peptides has beengenerated, they are then contacted with an appropriate amount of thepolymer substrate. The library of phage-peptides is dissolved in asuitable solution for contacting the substrate. The test substrate maybe suspended in the solution or may be immobilized on a plate or bead. Apreferred solution is a buffered aqueous saline solution containing asurfactant. A suitable solution is Tris-buffered saline (TBS) with 0.5%Tween® 20. The solution may additionally be agitated by any means inorder to increase the mass transfer rate of the peptides to the polymersubstrate, thereby shortening the time required to attain maximumbinding.

Upon contact, a number of the randomly generated phage-peptides willbind to the polymer substrate to form a phage-peptide-polymer complex.Unbound phage-peptide may be removed by washing. After all unboundmaterial is removed, phage-peptides having varying degrees of bindingaffinities for the polymer substrate may be fractionated by selectedwashings in buffers having varying stringencies. Increasing thestringency of the buffer used increases the required strength of thebond between the phage-peptide and polymer substrate in thephage-peptide-substrate complex.

A number of substances may be used to vary the stringency of the buffersolution in peptide selection including, but not limited to, acidic pH(1.5-3.0); basic pH (10-12.5); high salt concentrations such as MgCl₂(3-5 M) and LiCl (5-10 M); water; ethylene glycol (25-50%); dioxane(5-20%); thiocyanate (1-5 M); guanidine (2-5 M); urea (2-8 M); andvarious concentrations of different surfactants such as SDS (sodiumdodecyl sulfate), DOC (sodium deoxycholate), Nonidet P-40, Triton X-100,Tween® 20, wherein Tween® 20 is preferred. These substances may beprepared in buffer solutions including, but not limited to, Tris-HCl,Tris-buffered saline, Tris-borate, Tris-acetic acid, triethylamine,phosphate buffer, and glycine-HCl, wherein Tris-buffered saline solutionis preferred.

It will be appreciated that phage-peptides having increasing bindingaffinities for the PMMA substrate may be eluted by repeating theselection process using buffers with increasing stringencies. The elutedphage-peptides can be identified and sequenced by any means known in theart.

In one embodiment, the following method for generating the PMMA-bindingpeptides of the present invention may be used. A library ofcombinatorially generated phage-peptides is contacted with PMMA to formphage peptide-substrate complexes. The phage-peptide-substrate complexis separated from uncomplexed peptides and unbound substrate, and thebound phage-peptides from the phage-peptide-substrate complexes areeluted from the complex, preferably by acid treatment. Then, the elutedphage-peptides are identified and sequenced. To identify peptidesequences that bind to PMMA but not to other substrates, a subtractivepanning step may be added. Specifically, the library of combinatoriallygenerated phage-peptides is first contacted with the non-target toremove phage-peptides that bind to it. Then, the non-bindingphage-peptides are contacted with PMMA and the above process isfollowed. Alternatively, the library of combinatorially generatedphage-peptides may be contacted with the non-target and PMMAsimultaneously. Then, the phage-peptide-substrate complexes areseparated from the phage-peptide-non-target complexes and the methoddescribed above is followed for the desired phage-substrate complexes.

Alternatively, a modified phage display screening method for isolatingpeptides with a higher affinity for polymer substrates may be used. Inthe modified method, the phage-peptide-substrate complexes are formed asdescribed above. Then, these complexes are treated with an elutionbuffer. Any of the elution buffers described above may be used.Preferably, the elution buffer is an acidic solution. Then, theremaining, elution-resistant phage-peptide-substrate complexes are usedto directly infect/transfect a bacterial host cell, such as E. coliER2738. The infected host cells are grown in an appropriate growthmedium, such as LB (Luria-Bertani) medium, and this culture is spreadonto agar, containing a suitable growth medium, such as LB medium withIPTG (isopropyl β-D-thiogalactopyranoside) and S-Gal™. After growth, theplaques are picked for DNA isolation and sequencing to identify thepeptide sequences with a high binding affinity for the substrate ofinterest. Alternatively, PCR may be used to identify theelution-resistant phage-peptides from the modified phage displayscreening method, described above, by directly carrying out PCR on thephage-peptide-substrate complexes using the appropriate primers, asdescribed by Janssen et al. in U.S. Patent Application Publication No.2003/0152976, which is incorporated herein by reference.

Production of PMMA-Binding Peptides

The PMMA-binding peptides of the present invention may be prepared usingstandard peptide synthesis methods, which are well known in the art (seefor example Stewart et al., Solid Phase Peptide Synthesis, PierceChemical Co., Rockford, Ill., 1984; Bodanszky, Principles of PeptideSynthesis, Springer-Verlag, New York, 1984; and Pennington et al.,Peptide Synthesis Protocols, Humana Press, Totowa, N.J., 1994).Additionally, many companies offer custom peptide synthesis services.

Alternatively, the PMMA-binding peptides of the present invention may beprepared using recombinant DNA and molecular cloning techniques. Genesencoding the PMMA-binding peptides may be produced in heterologous hostcells, particularly in the cells of microbial hosts, as described byHuang et al. (U.S. Patent Application Publication No. 2005/0050656) andO'Brien et al., supra.

Preferred heterologous host cells for expression of the binding peptidesof the present invention are microbial hosts that can be found broadlywithin the fungal or bacterial families and which grow over a wide rangeof temperature, pH values, and solvent tolerances. Becausetranscription, translation, and the protein biosynthetic apparatus arethe same irrespective of the cellular feedstock, functional genes areexpressed irrespective of carbon feedstock used to generate cellularbiomass. Examples of host strains include, but are not limited to,fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces,Pichia, Candida, Hansenula, or bacterial species such as Salmonella,Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia,Pseudomonas, Methylomonas, Methylobacter, Alcaligenes, Synechocystis,Anabaena, Thiobacillus, Methanobacterium and Klebsiella.

A variety of expression systems can be used to produce the peptides ofthe present invention. Such vectors include, but are not limited to,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, frominsertion elements, from yeast episoms, from viruses such asbaculoviruses, retroviruses and vectors derived from combinationsthereof such as those derived from plasmid and bacteriophage geneticelements, such as cosmids and phagemids. The expression systemconstructs may contain regulatory regions that regulate as well asengender expression. In general, any system or vector suitable tomaintain, propagate or express polynucleotide or polypeptide in a hostcell may be used for expression in this regard. Microbial expressionsystems and expression vectors contain regulatory sequences that directhigh level expression of foreign proteins relative to the growth of thehost cell. Regulatory sequences are well known to those skilled in theart and examples include, but are not limited to, those which cause theexpression of a gene to be turned on or off in response to a chemical orphysical stimulus, including the presence of regulatory elements in thevector, for example, enhancer sequences. Any of these can be used toconstruct chimeric genes for production of the any of the bindingpeptides of the present invention. These chimeric genes can then beintroduced into appropriate microorganisms via transformation to providehigh level expression of the peptides.

Vectors or cassettes useful for the transformation of suitable hostcells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, one or more selectable markers, and sequences allowingautonomous replication or chromosomal integration. Suitable vectorscomprise a region 5′ of the gene, which harbors transcriptionalinitiation controls and a region 3′ of the DNA fragment which controlstranscriptional termination. It is most preferred when both controlregions are derived from genes homologous to the transformed host cell,although it is to be understood that such control regions need not bederived from the genes native to the specific species chosen as aproduction host. Selectable marker genes provide a phenotypic trait forselection of the transformed host cells such as tetracycline orampicillin resistance in E. coli.

Initiation control regions or promoters which are useful to driveexpression of the chimeric gene in the desired host cell are numerousand familiar to those skilled in the art. Virtually any promoter capableof driving the gene is suitable for producing the binding peptides ofthe present invention including, but not limited to: CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (usefulfor expression in Saccharomyces); AOX1 (useful for expression inPichia); and lac, ara, tet, trp, IP_(L), IP_(R), T7, tac, and trc(useful for expression in Escherichia coli) as well as the amy, apr, nprpromoters and various phage promoters useful for expression in Bacillus.

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary, however, it is most preferred if included.

The vector containing the appropriate DNA sequence as described supra,as well as an appropriate promoter or control sequence, may be employedto transform an appropriate host to permit the host to express thepeptide of the present invention. Cell-free translation systems can alsobe employed to produce such peptides using RNAs derived from the DNAconstructs of the present invention. Optionally, it may be desired toproduce the instant gene product as a secretion product of thetransformed host. Secretion of desired proteins into the growth mediahas the advantages of simplified and less costly purificationprocedures. It is well known in the art that secretion signal sequencesare often useful in facilitating the active transport of expressibleproteins across cell membranes. The creation of a transformed hostcapable of secretion may be accomplished by the incorporation of a DNAsequence that codes for a secretion signal which is functional in theproduction host. Methods for choosing appropriate signal sequences arewell known in the art (see for example EP 546049 and WO 9324631). Thesecretion signal DNA or facilitator may be located between theexpression-controlling DNA and the instant gene or gene fragment, and inthe same reading frame with the latter.

Active Domains

As noted above active domains are peptide portions of the PMMA bindingpeptide that convey various additional functionality to the peptide. Anysequence of amino acids may be used as an active domain, including, butnot limited to those functioning as a linker, those having bindingfunctionality, having catalytic functionality and those havingantimicrobial functionality.

An antimicrobial active domain may be particularly desirable if the PMMAmoiety part of the diblock was for instance part of a kitchen countertopsurface. Such antimicrobial sequences are well known in the art. Anypeptide based antimicrobial sequence could be used as an active domainin the above embodiment. As non-limiting examples table 1 providespossible antimicrobial active domain sequences.

TABLE 1 Antimicrobial active domain sequences. SEQ Species of ID originNO. Sequence Artificial 13 PKGLKKLLKGLKKLLKL Artificial 14KGLKKLLKGLKKLLKL Artificial 15 KGLKKLLKLLKKLLKL Artificial 16LKKLLKLLKKLLKL Artificial 17 LKKLLKLLKKLL Artificial 18VAKKLAKLAKKLAKLAL Artificial 19 FAKLLAKALKKLL Artificial 20KGLKKGLKLLKKLLKL Artificial 21 KGLKKLLKLGKKLLKL Artificial 22KGLKKLGKLLKKLLKL Artificial 23 KGLKKLLKLLKKGLKL Artificial 24KGLKKLLKLLKKLGKL Artificial 25 FALALKALKKLKKALKKAL Artificial 26FAKKLAKLAKKLAKLAL Artificial 27 FAKLLAKLAKKLL Artificial 28FAKKLAKLALKLAKL Artificial 29 FAKKLAKKLL Artificial 30 FAKLLAKLAKKVLArtificial 31 KYKKALKKLAKLL Artificial 32 FALLKALLKKAL Artificial 33KRLFKKLKFSLRKY Artificial 34 KRLFKKLLFSLRKY Artificial 35 LLLFLLKKRKKRKYH. cecropia 36 KWKLFKKIEKVGQNIRDGIIKAGPAVAWGQATQIAK Xenopus 37GIGKFLHSAKKFGKAFVGEIMNS Xenopus 38 GIGKFLKKAKKFGKAFVKILKK Bos Taurus 39RLCRIVVIRVCR Bos Sp. 40 ILPWKWPWWPWRR H. sapiens 41DSHAKRHHGYKRKFHEKHHSHRGY

Two sub-types of active domains, target binding domains and linkingdomains, have been given specific names in the discussion of thispresent invention. A target binding domain is an active domain thatspecifically binds to a known target. Target binding sequences are knownin the art and can be developed using known techniques as well astechniques described herein. Non-limiting examples of targets to whichtarget binding domains will bind include, pigments, dyes, chemicalfunctional groups, print media, body surfaces (hair, skin, nails, teethetc.) and biological analytes (cells, receptors, proteins, nucleicacids, viral particles, prions etc.) (see FIGS. 4, 5 and 6 panel D).

A linking domain is an active domain that is specifically used toseparate two domains or a domain from a terminal end. Any sequence ofamino acids that does not contain a PMMA-binding site can be used as alinking domain. A linking domain can have activity beyond justseparating two features of a peptide. A linking domain may provide aspecific structure to the separating portion of the peptide. Conversely,a linking domain may also be selected to provide flexibility to theseparating portion of the peptide. Additionally the linking domain maybe created to specifically change the rheology of the medium the peptideis immersed in. Also the linking domain may be constructed so that itcan be cleaved by, or act as the binding site for, a cleaving moleculeor enzyme, for the purpose of releasing a portion of the peptide and/orthe PMMA from the complex.

Preferred peptide linker domains are composed of the amino acidsproline, lysine, glycine, alanine, and serine, and mixtures thereof. Inaddition, the peptide linker may contain a specific enzyme cleavagesite, such as the protease Caspase 3 site, given by SEQ ID NO:176, whichallows for the enzymatic removal of a portion of the peptide and/or thePMMA from the complex. The peptide linker may be from 1 to about 50amino acids, preferably from 1 to about 20 amino acids in length.Examples of peptide linkers include, but are not limited to, SEQ IDNOs:176 to 179. These peptide linkers may be linked to the bindingpeptide sequence by any method know in the art. For example, the entirebinding peptide-peptide linker-diblock may be prepared using thestandard peptide synthesis methods described supra. In addition, thebinding peptide and peptide linker blocks may be combined usingcarbodiimide coupling agents (see for example, Hermanson, BioconjugateTechniques, Academic Press, New York (1996)), diacid chlorides,diisocyanates and other difunctional coupling reagents that are reactiveto terminal amine and/or carboxylic acid groups on the peptides.Alternatively, the entire binding peptide-peptide linker-diblock may beprepared using the recombinant DNA and molecular cloning techniquesdescribed supra. The linker may also be a combination of a peptidelinker and an organic linker molecule, which may be prepared using themethods described above. Examples of specific linker peptides are givenin table 2 below.

TABLE 2 Linker peptides Species of SEQ ID origin NO. Sequence Artificial176 LESGDEVD Artificial 177 TSTSKASTTT TSSKTTTTSS KTTTTTSKTS TTSSSSTArtificial 178 GQGGYGGLGS QGAGRGGLGG QG Artificial 179 GPGGYGPGQQ

Target domains of the invention are another type of active domaincomprised within the PMMA binding peptide. Target domains will havebinding affinity for various substance such as benefit agents (pigments,dyes, print media, biological analtyes, body surfaces (hair, skin,nails, teeth etc.) and the like).

Pigment binding domains are target domains that bind various pigmentsand colorants. Such pigments have application in the personal care aswell as the printing industries. Similarly print media binding domainsare target binding domains having specific affinity for various types ofprint media. Typically the print media will comprise cotton or cellulosetargets or may be coated with a polymer such as nylon or PMMA givingrise to cotton, cellulose or polymer binding domains as part of the PMMAbinding peptide.

Target domains may be uni-functional having binding affinity for asingle target species or multifunctional, having affinity for a varietyof targets. For example it may be desirable to combine a pigment bindingdomain or a print medium binding domain or both into the peptide part ofthe PMMA-peptide complex of the present invention. Such an embodimentthat includes a print-medium binding domain may be particularlydesirable if the complex already contains a benefit agent that is acolorant or dye. Pigment-binding peptides and print medium-bindingpeptides have been identified (See tables 3, 4, and 5, and O'Brien etal., supra, hereby incorporated by reference. The pigment-bindingpeptides typically comprise at least about 40 mole % of the amino acids:glycine, alanine, valine, leucine, isoleucine, methionine, proline,phenylalanine, and tryptophan Specifically, binding peptides wereisolated that have a high affinity for the pigments carbon black, givenas SEQ ID NOs:42-45, CROMOPHTHAL® Yellow, given as SEQ ID NOs: 46-53,SUNFAST® Magenta, given as SEQ ID NOs: 55-57, and SUNFAST® Blue, givenas SEQ ID NOs: 54, 58-66. The cellulose-binding peptides of theinvention comprise at least about 14 mole % of the amino acids: serine,threonine and tyrosine. Binding peptides having a high binding affinityfor cellulose (a major component of cotton) include SEQ ID NOs: 73-78.The polyester-binding peptides of the invention comprise at least about20 mole % of the amino acids: phenylalanine, tryptophan, and tyrosine.Binding peptides having a high affinity for polyester (poly(ethyleneterephthalate)) include SEQ ID NO: 79. Additionally, binding peptideswere isolated that have a binding affinity for the following printmedia: cotton, given as SEQ ID NOs: 67 and 68, polyester/cotton, givenas SEQ ID NOs: 67 and 69, and printing paper, given as SEQ ID NOs: 67,and 70-72.

TABLE 3 Pigment-Binding Peptides Designated Peptide SEQ ID Pigment NameSequence NO: Carbon Black CB-71 MPPPLMQ 42 CB-72 FHENWPS 43 CB-121RTAPTTPLLLSL 44 CR-122 WHLSWSPVPLPT 45 Cromophtal ® Yellow CY-71 PHARLVG46 CY-72 NIPYHHP 47 CY-73 TTMPAIP 48 CY-74 HNLPPRS 49 CY-121AHKTQMGVRQPA 50 CY-122* ADNVQMGVSHTP 51 CY-123* AHNAQMGVSHPP 52 CY-124*ADYVGMGVSHRP 53 CY-125 SVSVGMKPSPRP 54 Sunfast ® Magenta SM-71 YPNTALV55 SM-72 VATRIVS 56 SM-121 HSLKNSMLTVMA 57 Sunfast ® Blue SB-71 NYPTQAP58 SB-72 KCCYSVG 59 SB-121 RHDLNTWLPPVK 60 SB-122 EISLPAKLPSAS 61 SB-123SVSVGMKPSPRP 54 SB-124** SDYVGMRPSPRH 62 SB-125** SDYVGMRLSPSQ 63SB-126** SVSVGIQPSPRP 64 SB-127** YVSVGIKPSPRP 65 SB-128** YVCEGIHPCPRP66 *These sequences are analogs of CY-121. **These sequences are eitheranalogs of SB-123 or are similar to the analogs of SB-123.

TABLE 4 Print Medium-Binding Peptides Designated Peptide SEQ IDPrint Medium Name Sequence NO: Cotton fabric COT-71* SILPYPY 67 COT-72STASYTR 68 Polyester/cotton fabric P/C-71 LPVRPWT 69 P/C-72* SILPYPY 67Hammermill ® paper HCP-71 GNTPSRA 70 HCP-72 HAIYPRH 71 HCP-73 YQDSAKT 72HCP-74* SILPYPY 67 *These sequences are identical.

TABLE 5 Cellulose and Poly(ethylene terephthalate)- Binding PeptidesPrint Medium Designated Peptide Ingredient Name Sequence SEQ ID NO:Cellulose CEL-71 VPRVTSI 73 CEL-72 MANHNLS 74 CEL-73 FHENWPS 75 CEL-121THKTSTQRLLAA 76 CEL-122 KCCYVNVGSVFS 77 CEL-123 AHMQFRTSLTPH 78Poly(ethylene PET-121 GTSDHMIMPFFN 79 terephthalate)

Target domains that have binding affinity for body surfaces areparticularly useful for the production of personal care compositionscomprising colorants, and conditioners with specific binding affinityfor the body surface. For example, it may be desirable to attachPMMA-peptide complex of the present invention in either a tri-block formor a di-block form to a body surface such as hair or skin. One method toachieve such a result is to incorporate a target binding domain into thepeptide part of the present invention that binds hair, skin or anotherbody surface. Both hair and skin binding domains can be produced by themethods described here, in the co-pending, commonly owned U.S. Ser. No.10/935,642 (U.S. Patent Application Publication No. 2005/0050656) herebyincorporated by reference and in co-pending, commonly owned U.S. Ser.No. 11/074,473 (U.S. Patent Application Publication No. 2005/0226839)also hereby incorporated by reference. Examples of hair and skin bindingdomains are shown in Table 6.

TABLE 6 Body Surface Binding Peptide Domains SEQ Body ID Surface NOSequence Skin 80 FTQSLPR Skin 81 TPFHSPENAPGS Skin 82 KQATFPPNPTAY Skin83 HGHMVSTSQLSI Skin 84 LSPSRMK Skin 85 LPIPRMK Skin 86 HQRPYLT Skin 87FPPLLRL Nail 88 ALPRIANTWSPS Nail 89 YPSFSPTYRPAF Hair 90 YPSFSPTYRPAFHair 91 ALPRIANTWSPS Hair 92 LESTPKMK Hair 93 SVSVGMKPSPRP Hair 94LDVESYKGTSMP Hair 95 RVPNKTVTVDGA Hair 96 DRHKSKYSSTKS Hair 97KNFPQQKEFPLS Hair 98 QRNSPPAMSRRD Hair 99 TRKPNMPHGQYL Hair 100KPPHLAKLPFTT Hair 101 NKRPPTSHRIHA Hair 102 NLPRYQPPCKPL Hair 103RPPWKKPIPPSE Hair 104 RQRPKDHFFSRP Hair 105 SVPNK(T or P)VTVDGX Hair 106TTKWRHRAPVSP Hair 107 WLGKNRIKPRAS Hair 108 SNFKTPLPLTQS Hair 109KELQTRNVVQRE Hair 110 GMPAMHWIHPFA Hair 111 TPTANQFTQSVP Hair 112AAGLSQKHERNR Hair 113 ETVHQTPLSDRP Hair 114 LPALHIQRHPRM Hair 115QPSHSQSHNLRS Hair 116 RGSQKSKPPRPP Hair 117 THTQKTPLLYYH Hair 118TKGSSQAILKST Hair 119 DLHTVYH Hair 120 HIKPPTR Hair 121 HPVWPAI Hair 122MPLYYLQ Hair 123 HLTVPWRGGGSAVPFYSHSQITLPNH Hair 124GPHDTSSGGVRPNLHHTSKKEKRENRKVPFYSHSVTS RGNV Hair 125 KHPTYRQ Hair 126HPMSAPR Hair 127 MPKYYLQ Hair 128 MHAHSIA Hair 129 TAATTSP Hair 130LGIPQNL Hair 131 AKPISQHLQRGS Hair 132 APPTPAAASATT Hair 133DPTEGARRTIMT Hair 134 EQISGSLVAAPW Hair 135 LDTSFPPVPFHA Hair 136LPRIANTWSPS Hair 137 RTNAADHPAAVT Hair 138 SLNWVTIPGPKI Hair 139TDMQAPTKSYSN Hair 140 TIMTKSPSLSCG Hair 141 TPALDGLRQPLR Hair 142TYPASRLPLLAP Hair 143 AKTHKHPAPSYS Hair 144 TDPTPFSISPER Hair 145CAAGCCTCAGCGACCGAATA Hair 146 WHDKPQNSSKST Hair 147 NEVPARNAPWLV Hair148 NSPGYQADSVAIG Hair 149 TQDSAQKSPSPL Hair 150 TPPELLHGDPRS Hair 151TPPTNVLMLATK Hair 152 NTSQLST Hair 153 NTPKENW Hair 154 NTPASNR Hair 155PRGMLST Hair 156 PPTYLST Hair 157 TIPTHRQHDYRS Hair 158 TPPTHRL Hair 159LPTMSTP Hair 160 LGTNSTP Hair 161 TPLTGSTNLLSS Hair 162 TPLTKET Hair 163QQSHNPP Hair 164 TQPHNPP Hair 165 STNLLRTSTVHP Hair 166 HTQPSYSSTNLFHair 167 SLLSSHA Hair 168 QQSSISLSSHAV Hair 169 NASPSSL Hair 170 HSPSSLRHair 171 K(H, R or N)SHHTH Hair 172 E(H, R, or N)SHHTH Hair 173 LESTSLLHair 174 TPLTKET Hair 175 KQSHNPP

If the present invention is desired to be used in connection with a haircare composition an effective amount of the complex for use in a haircare composition is herein defined as a proportion of from about 0.01%to about 10%, preferably about 0.01% to about 5% by weight relative tothe total weight of the composition. Components of a cosmeticallyacceptable medium for hair care compositions are described by Philippeet al. in U.S. Pat. No. 6,280,747, and by Omura et al. in U.S. Pat. No.6,139,851 and Cannell et al. in U.S. Pat. No. 6,013,250, all of whichare incorporated herein by reference. For example, these hair carecompositions can be aqueous, alcoholic or aqueous-alcoholic solutions,the alcohol preferably being ethanol or isopropanol, in a proportion offrom about 1 to about 75% by weight relative to the total weight, forthe aqueous-alcoholic solutions. Additionally, the hare carecompositions may contain one or more conventional cosmetic ordermatological additives or adjuvants including but not limited to,antioxidants, preserving agents, fillers, surfactants, UVA and/or UVBsunscreens, fragrances, thickeners, wetting agents and anionic, nonionicor amphoteric polymers, and dyes or pigments.

In a number of embodiments the present invention could be used in a skincare composition. Skin care compositions are herein defined ascompositions comprising an effective amount of a skin conditioner or amixture of different skin conditioners in a cosmetically acceptablemedium. The uses of these compositions include, but are not limited to,skin care, skin cleansing, make-up, and anti-wrinkle products. If thepresent invention is desired to be used in connection with a skin carecomposition an effective amount of the complex for skin carecompositions is herein defined as a proportion of from about 0.001% toabout 10%, preferably about 0.01% to about 5% by weight relative to thetotal weight of the composition. This proportion may vary as a functionof the type of skin care composition. Suitable compositions for acosmetically acceptable medium are described by Philippe et al. supra.For example, the cosmetically acceptable medium may be an anhydrouscomposition containing a fatty substance in a proportion generally offrom about 10 to about 90% by weight relative to the total weight of thecomposition, where the fatty phase containing at least one liquid, solidor semi-solid fatty substance. The fatty substance includes, but is notlimited to, oils, waxes, gums, and so-called pasty fatty substances.Alternatively, the compositions may be in the form of a stabledispersion such as a water-in-oil or oil-in-water emulsion.Additionally, the compositions may contain one or more conventionalcosmetic or dermatological additives or adjuvants, including but notlimited to, antioxidants, preserving agents, fillers, surfactants, UVAand/or UVB sunscreens, fragrances, thickeners, wetting agents andanionic, nonionic or amphoteric polymers, and dyes or pigments.

Benefit Agents

Benefit agents are any material or substance that may be complexed withthe PMMA binding peptide in an manner so as to deliver a benefit at thepoint where the PMMA binding peptide is attached. In the most generalsense the benefit agent will be the third component of the tri-block ofPMMA and PMMA binding peptide. Any complex, compound or element may beused with the present invention as a benefit agent. If a user of theinvention desires to have the features of a benefit agent combined withPMMA then a triblock may be constructed to include the benefit agent inthe formation with PMMA and a PMMA-binding peptide. A benefit agent maybe selected for the purpose of adding the physical, chemical and/orbiological properties of said agent to the PMMA-peptide complex of thepresent invention. The result of this construct will be said benefitagent closely associated with PMMA and the activity of said benefitagent will be included within the triblock.

The triblock embodiment of this present invention is composed of atleast one member of each block element but may also have multiple copiesof identical or different members of one, two or all three blockelements. Benefit agents can be used singularly or in a plurality. Insome embodiments a plurality of peptide blocks or a plurality of PMMAblocks or a plurality of both blocks may be added to a single benefitagent block or a number of benefit agent blocks. For some small benefitagents, for non-limited example, those composed of an element, as manyas 10,000 benefit agents could be added to a single PMMA-complex. Forsome large benefit agents, for non-limiting example, a dye embedded in aplastic bead as many as 10,000 PMMA complexes might be attached to asingle benefit agent.

Benefit agents may be inorganic or organic in nature, this includesbeing polymer or peptide based. They may not be by definition eithercomposed of PMMA or part of a PMMA-binding peptide, since suchcompositions are defined as categorically other parts of the triblockformation. Some preferred embodiments include benefit agents that arepigments, pharmaceuticals, markers, conditioners, colorants, andfragrances.

Pharmaceuticals.

A pharmaceutical generally means a substance dosed to an organism orthing to treat or prevent a disease or condition. A pharmaceuticalbenefit agent includes, in a non-limiting sense, the topical, internalor intracellular administration of a compound to an organism as atreatment or a prophylactic. A non-limiting example of this embodimentof the present invention would be the attachment of an anti-acnemedication to formulation of the present invention designed to be a skinconditioner. A pharmaceutical benefit agent also includes a treatment tosurface or item to prevent an infectious germ from being transmittedafter contacting said surface or item. The addition of an antimicrobialcompound to a construction of the present invention to be used oncountertops would be a non-limiting example of this embodiment. Suitablepharmaceuticals are well known in the art. An extensive list is given byKabonov et al. in U.S. Pat. No. 6,696,089, which is incorporated hereinby reference (in particular, columns 16 to 18). Examples include, butare not limited to, antibacterial agents, antiviral agents, antifungalagents, anti-cancer agents, vaccines, radiolabels, anti-inflammatories,anti-glaucomic agents, local anesthetics, anti-neoplastic agents,antibodies, hormones, and the like.

Markers.

Markers as used and defined herein refer to a class of benefit agentsthat provide aid in detecting the presence of the PMMA-peptide complexto which they are, or were, attached. The marker benefit agent might bea dye, fluorescent label, radioactive element or some other signal.Radioactive P³² is a non-limiting example of this type of marker benefitagent. Also the marker benefit agent might also be a substance thatreacts with a dye, fluorescent label or other signal. Biotin used inconnection with a labled-streptavidin compound is a non-limited exampleof this type of marker benefit agent. Additionally a marker benefitagent might also provide, or help to provide aid to detect, the presenceor lack of presence of another specific chemical, compound, element orcomplex. By way of non-limiting example, the marker benefit agent mightbe a compound that is metabolized by a specific enzyme to produce ametabolite that reacts with a fluorescently labeled phosphine. TheStaudinger ligation is a non-limiting example of this type of markerbenefit agent.

Conditioners.

Conditioner benefits agents as referred to in discussion of the presentinvention generally mean benefit agents that provide an improvement tothe appearance, texture or quality of the substance they are designed tocondition. Conditioner benefit agents may be used with the presentinvention to condition any substance including but not limited to hair,skin, lips, leather, and upholstery. In the preferred embodiment thepresent invention is used in combination with a benefit agent thatprovides a conditioning effect to hair and skin. In the most preferredembodiment said hair and skin are human hair and human skin.

Hair conditioning agents as herein defined are agents which improve theappearance, texture, and sheen of hair as well as increasing hair bodyor suppleness. In the peptide-based hair conditioners of the presentinvention, any known hair conditioning agent may be used. Hairconditioning agents are well known in the art, see for example Green etal. (WO 0107009), incorporated herein by reference, and are availablecommercially from various sources. Suitable examples of hairconditioning agents include, but are not limited to, cationic polymers,such as cationized guar gum, diallyly quaternary ammoniumsalt/acrylamide copolymers, quaternized polyvinylpyrrolidone andderivatives thereof, and various polyquaternium-compounds; cationicsurfactants, such as stearalkonium chloride, centrimonium chloride, andSapamin hydrochloride; fatty alcohols, such as behenyl alcohol; fattyamines, such as stearyl amine; waxes; esters; nonionic polymers, such aspolyvinylpyrrolidone, polyvinyl alcohol, and polyethylene glycol;silicones; siloxanes, such as decamethylcyclopentasiloxane; polymeremulsions, such as amodimethicone; and volumizing agents, such asnanoparticles (e.g., silica nanoparticles and polymer nanoparticles).The preferred hair conditioning agents of the present invention containamine or hydroxyl functional groups to facilitate coupling to thehair-binding peptides. Examples of preferred conditioning agents areoctylamine (CAS No. 111-86-4), stearyl amine (CAS No. 124-30-1), behenylalcohol (CAS No. 661-19-8, Cognis Corp., Cincinnati, Ohio), vinyl groupterminated siloxanes, vinyl group terminated silicone (CAS No.68083-19-2), vinyl group terminated methyl vinyl siloxanes, vinyl groupterminated methyl vinyl silicone (CAS No. 68951-99-5), hydroxylterminated siloxanes, hydroxyl terminated silicone (CAS No. 80801-30-5),amino-modified silicone derivatives, [(aminoethyl)amino]propyl hydroxyldimethyl siloxanes, [(aminoethyl)amino]propyl hydroxyl dimethylsilicones, and alpha-tridecyl-omega-hydroxy-poly(oxy-1,2-ethanediyl)(CAS No. 24938-91-8).

Skin conditioning agents as herein defined include, but are not limitedto astringents, which tighten skin; exfoliants, which remove dead skincells; emollients, which help maintain a smooth, soft, pliableappearance; humectants, which increase the water content of the toplayer of skin; occlusives, which retard evaporation of water from theskin's surface; and miscellaneous compounds that enhance the appearanceof dry or damaged skin or reduce flaking and restore suppleness. In thepeptide-based skin conditioners of the present invention, any known skinconditioning agent may be used. Skin conditioning agents are well knownin the art, see for example Green et al. supra, and are availablecommercially from various sources. Suitable examples of skinconditioning agents include, but are not limited to, alpha-hydroxyacids, beta-hydroxy acids, polyols, hyaluronic acid, D,L-panthenol,polysalicylates, vitamin A palmitate, vitamin E acetate, glycerin,sorbitol, silicones, silicone derivatives, lanolin, natural oils andtriglyceride esters. The preferred skin conditioning agents of thepresent invention are polysalicylates, propylene glycol (CAS No.57-55-6, Dow Chemical, Midland, Mich.), glycerin (CAS No. 56-81-5,Proctor & Gamble Co., Cincinnati, Ohio), glycolic acid (CAS No. 79-14-1,DuPont Co., Wilmington, Del.), lactic acid (CAS No. 50-21-5, Alfa Aesar,Ward Hill, Mass.), malic acid (CAS No. 617-48-1, Alfa Aesar), citricacid (CAS No. 77-92-9, Alfa Aesar), tartaric acid (CAS NO. 133-37-9,Alfa Aesar), glucaric acid (CAS No. 87-73-0), galactaric acid (CAS No.526-99-8), 3-hydroxyvaleric acid (CAS No. 10237-77-1), salicylic acid(CAS No. 69-72-7, Alfa Aesar), and 1,3 propanediol (CAS No. 504-63-2,DuPont Co., Wilmington, Del.). Polysalicylates may be prepared by themethod described by White et al. in U.S. Pat. No. 4,855,483,incorporated herein by reference. Glucaric acid may be synthesized usingthe method described by Merbouh et al. (Carbohydr. Res. 336:75-78(2001). The 3-hydroxyvaleric acid may be prepared as described byBramucci in WO 02012530.

Colorants.

The term colorant generally refers to a coloring agent. Colorants may bechemically organic or inorganic and may include pigments or dyes. Thepeptide-based colorants of the present invention may be prepared bycovalently attaching a specific PMMA-binding peptide to a coloringagent, either directly or via a linker, using any of the couplingmethods known in the art (see for example, U.S. Patent ApplicationPublication No. 2005/0226839).

Pigments are a particularly suitable benefit agent. Pigments generallymeans an insoluble colorant. A wide variety of organic and inorganicpigments alone or in combination may be used in the present invention.Examples of organic pigments include, but are not limited to Cyan,Yellow, Red, Blue, Orange, Magenta, Black, Green, Violet, Light Cyan,and Light Magenta. Preferred organic pigments are carbon black, such asCarbon Black FW18, and colored pigments such as CROMOPHTHAL® Yellow131AK (Ciba Specialty Chemicals), SUNFAST® Magenta 122 (Sun Chemical)and SUNFAST® Blue 15:3 (Sun Chemical). Examples of inorganic pigmentsinclude, but are not limited to finely divided metals, such as copper,iron, aluminum, and alloys thereof; and metal oxides, such as silica,alumina, and titania. Additional examples of suitable pigments are givenby Ma et al. in U.S. Pat. No. 5,085,698, incorporated herein byreference.

The preferred coloring agents for use in the skin based applications ofthe present invention include but are not limited to the following dyes:eosin derivatives such as D&C Red No. 21 and halogenated fluoresceinderivatives such as D&C Red No. 27, D&C Red Orange No. 5 in combinationwith D&C Red No. 21 and D&C Orange No. 10, and the pigments: titaniumdioxide, zinc oxide, D&C Red No. 36 and D&C Orange No. 17, the calciumlakes of D&C Red Nos. 7, 11, 31 and 34, the barium lake of D&C Red No.12, the strontium lake D&C Red No. 13, the aluminum lakes of FD&C YellowNo. 5, of FD&C Yellow No. 6, of D&C Red No. 27, of D&C Red No. 21, ofFD&C Blue No. 1, iron oxides, manganese violet, chromium oxide,ultramarine blue, and carbon black.

The preferred coloring agents for use with the present invention in thenail based applications include but are not limited to D&C Red Nos. 8,10, 30 and 36, the barium lakes of D&C Red Nos. 6, 9 and 12, the calciumlakes of D&C Red Nos. 7, 11, 31 and 34, the strontium lake of D&C RedNo. 30 and D&C Orange No. 17 and D&C Blue No. 6. Suitable hair coloringagents for use with the present invention include, but are not limitedto dyes, such as 4-hydroxypropylamino-3-nitrophenol,4-amino-3-nitrophenol, 2-amino-6-chloro-4-nitrophenol,2-nitro-paraphenylenediamine, N,N-hydroxyethyl-2-nitro-phenylenediamine,4-nitro-indole, Henna, HC Blue 1, HC Blue 2, HC Yellow 4, HC Red 3, HCRed 5, Disperse Violet 4, Disperse Black 9, HC Blue 7, HC Blue 12, HCYellow 2, HC Yellow 6, HC Yellow 8, HC Yellow 12, HC Brown 2, D&C Yellow1, D&C Yellow 3, D&C Blue 1, Disperse Blue 3, Disperse violet 1, eosinderivatives such as D&C Red No. 21 and halogenated fluoresceinderivatives such as D&C Red No. 27, D&C Red Orange No. 5 in combinationwith D&C Red No. 21 and D&C Orange No. 10; and pigments, such as D&C RedNo. 36 and D&C Orange No. 17, the calcium lakes of D&C Red Nos. 7, 11,31 and 34, the barium lake of D&C Red No. 12, the strontium lake of D&CRed No. 13, the aluminum lakes of FD&C Yellow No. 5, of FD&C Yellow No.6, of D&C Red No. 27, of D&C Red No. 21, and of FD&C Blue No. 1, ironoxides, manganese violet, chromium oxide, titanium dioxide, iron oxides,zinc oxide, barium oxide, ultramarine blue, bismuth citrate, and carbonblack particles.

The preferred hair coloring agents of the present invention are D&CYellow 1 and 3, HC Yellow 6 and 8, D&C Blue 1, HC Blue 1, HC Brown 2, HCRed 5,2-nitro-paraphenylenediamine,N,N-hydroxyethyl-2-nitro-phenylenediamine, 4-nitro-indole, iron oxides,and carbon black.

Fragrances.

A fragrance is a complex, compound or element that releases, a substancewhich may be perceived by the sense of olfaction or chemical detectionin any organism, but preferably, in humans. The object sensed ordetected may be a part of or the whole of the fragrance benefit agent.In the preferred embodiment the odor is perceived as desirable tohumans. However, some uses may combine the present invention with afragrance benefit agent that is repellent to a class of organisms,including a class that contains or is humans. Any known fragrance orodor may be use as a benefit agent. It may be desirable to attach afragrance benefit agent to the PMMA-peptide complex by a bond structureor linking molecule that allows the benefit agent to be released, inpart or in whole, so that it may be perceived by a sensing organ orchemical detector.

Numerous fragrances, both natural and synthetic, are well known in theart. For example, Secondini (Handbook of Perfumes and Flavors, ChemicalPublishing Co., Inc., New York, 1990), incorporated herein by reference,describes many of the natural and synthetic fragrances used incosmetics. Suitable natural fragrances include, but are not limited, tojasmines, narcissus, rose, violet, lavender, mint, spice, vanilla,anise, amber, orange, pine, lemon, wintergreen, rosemary, basil, andspruce. Suitable synthetic fragrances include, but are no limited to,acetaldehyde, C7 to C16 alcohols, benzyl acetate, butyric acid, citricacid, isobutyl phenyl acetate, linalyl butyrate, malic acid, menthol,phenyl ethyl cinnamate, phenyl propyl formate, tannic acid, terpineol,vanillin, amyl salicylate, benzaldehyde, diphenyl ketone, indole, andthe like.

Linker Molecules

Linker molecules may optionally be used with some embodiments of thepresent invention for the purpose of attaching the benefit agent to thePMMA-peptide complex (see FIG. 3, reference number 225). Any molecule,compound or complex that will attach the benefit agent to the complexcan be used as a linking molecule provided the linking molecule does notcontain PMMA or a PMMA-binding domain. The benefit agent may be attachedto the complex to either the PMMA moiety or the peptide portion or inthe case of a plurality of benefit agent possibly to both. The linkingmolecules may be designed to bond the benefit agent stably or in thealternative they may be designed to break and release the benefit agentfrom the complex in a given circumstance. Such circumstances could be,for non-limiting example, a range of pH, a range of temperatures, arange of pressure, while immersed in a certain media, the presence of aparticular element, molecule or compound at a certain range ofconcentration, after a given passage of time, or at a certain averagerate for a population of linker molecules.

Specifically the linker may be any of a variety of molecules, such asalkyl chains, phenyl compounds, ethylene glycol, amides, esters and thelike. Preferred linkers are hydrophilic and have a chain length from 1to about 100 atoms, more preferably, from 2 to about 30 atoms. Examplesof preferred linkers include, but are not limited to, ethanol amine,ethylene glycol, polyethylene with a chain length of 6 carbon atoms,polyethylene glycol with 3 to 6 repeating units, phenoxyethanol,propanolamide, butylene glycol, butyleneglycolamide, propyl phenyl, andethyl, propyl, hexyl, steryl, cetyl, and palmitoyl alkyl chains. Thelinker may be covalently attached to the peptide and the benefit agentusing any of the coupling chemistries described above. In order tofacilitate incorporation of the linker, a bifunctional cross-linkingagent that contains a linker and reactive groups at both ends forcoupling to the peptide and the benefit agent may be used. Suitablebifunctional cross-linking agents are well known in the art and include,but are not limited to diamines, such as 1,6-diaminohexane; dialdehydes,such as glutaraldehyde; bis N-hydroxysuccinimide esters, such asethylene glycol-bis(succinic acid N-hydroxysuccinimide ester),disuccinimidyl glutarate, disuccinimidyl suberate, and ethyleneglycol-bis(succinimidylsuccinate); diisocyantes, such ashexamethylenediisocyanate; bis oxiranes, such as 1,4 butanediyldiglycidyl ether; dicarboxylic acids, such as succinyldisalicylate; andthe like. Heterobifunctional cross-linking agents, which contain adifferent reactive group at each end, may also be used.

Applications of PMMA Binding Peptides

It will be appreciated by the skilled person that PMMA binding peptidescomprising active and target domains having specific functionality maybe used in a multiplicity of formats including as delivery means fordelivering benefits agents, in assays for diagnostic applications aswell as in materials applications for coating PMMA surfaces. Thefollowing description of the figures presents a limited number ofexamples of the method of the invention, but is by no means inclusive ofall possible applications and formats.

Referring to FIG. 1 panel A, there is shown a surface 1 comprising, inwhole or in part, a PMMA moiety 3. At least some of the PMMA molecules 3are exposed in various orientations on the exterior of the surface. ThePMMA binding peptide 5 comprises at least one, but not limited to one,PMMA binding domain 7. The PMMA binding domain 7 further comprises atleast one, but not limited to one, PMMA binding site 15. PMMA bindingpeptides 5 will bind specifically to PMMA molecules 3, this binding willoccur at the PMMA binding site 15 of the PMMA domain 7 within the PMMAbinding peptide 5. The PMMA-binding peptide 5 coupled to the PMMA moiety3 forms a diblock structure. The formation of this diblock structure ona PMMA containing surface 1, such as CORIAN® is useful for coating suchsurfaces with a protein layer to serve as a sacrificial layer or to maskproperties of the surface 1.

FIG. 1 panel B depicts another embodiment of the invention. In thisembodiment, a PMMA binding peptide 5 also binds to a surface 1comprising, in whole or in part, PMMA molecules 3. A benefit agent 19 iscoupled to the PMMA binding peptide 5 covalently, ionically or otherwiseas described elsewhere herein. Although bound to the PMMA bindingpeptide 5, the benefit agent 19 generally retains the biological,chemical and physical properties that it exhibited before being coupledto the PMMA binding peptide 5. The complex of the PMMA particle 3, thePMMA-binding peptide 5, and the benefit agent 19 forms a triblock. Theproximity of the benefit agent 19 to the surface 1 after binding allowsthe benefit agent 19 to be active at that location, and provides thechemical property of the benefit agent 19 on the PMMA containing surface1. Non-limiting examples of the benefit agents 19 are colorants such asdyes and pigments, conditioners, fragrances, pharmaceuticals and thelike.

FIG. 1 panel C depicts still another embodiment of the invention. ThePMMA binding peptide 5 binds to a surface 1 comprising PMMA 3 as above.Panel C, as in panels A and B, shows the PMMA binding peptide 5comprising at least one, but not limited to one, PMMA binding domain 7within its structure. The PMMA binding domain 7 comprises at least one,but not limited to one, PMMA binding site 15. The PMMA binding peptide 5of panel C further comprises at least one, but not limited to one,active domain 9 different from the PMMA binding domain 7, yet within thesame PMMA binding peptide 5. By having an active domain 9 within thepeptide 5 and the peptide 5 being bound to a PMMA-containing surface 1this embodiment of the invention allows the property of the activedomain 9 to be transmitted to the surface 1. One non-limiting example ofan active domain as exemplified here is a domain having antimicrobialproperties.

FIG. 1 panel D depicts still another embodiment of the invention. ThePMMA binding peptide 5 binds to a surface 1 comprising PMMA 3 asdescribed above. In this embodiment, the PMMA binding peptide 5comprises a specific target binding domain 11 targeting other moleculesother than PMMA. In this embodiment, the PMMA-binding peptide 1 acts asan intermediary to bring the target molecules close to the surface 1.This may be used to provide the chemical, biologic or physical functionof target molecule 17 on the surface 1. However, this embodiment mayalso be employed to isolate the target molecule 17 from the surroundingmedia. Another use may be to sample the surrounding media for thepresence of the target molecule 17. Non-limiting examples of the othertarget molecule 17 include benefit agents such as colorants (dyes andpigments) and conditioners as well as biological analytes, (cells,membrane fractions, viral particles, proteins, nucleic acids and thelike), body surfaces, (hair, skin, nails, teeth and the like) as well asother organic and inorganic target complexes.

FIG. 1 panel E depicts still another embodiment of the invention. ThePMMA binding peptide 5 binds to a surface 1 comprising PMMA 3 as above.Panel E depicts a PMMA binding peptide 5 that contains a linker domain13 that serves to connect the PMMA binding peptide 5 to a benefit agent19. The linker domain 13 is a domain that selected to physicallyseparate the benefit agent 19 from the PMMA binding domain(s) 7.Alternatively, although not depicted in the FIG. 1, a single linkerdomain or many linker domains may be provided to separate variousdomains within the PMMA-binding peptide. For instance, it may beadvantageous to separate the PMMA binding domain from an active domain,or to separate two or more active domains, a linker could be utilized toachieve this separation. The linker domain 13 may simply provide asteric benefit. Although in some uses of this embodiment the linkerprovides a specific structure or orientation between the PMMA bindingpeptide 5 and the benefit agent 19 or to limit the conformation of thebenefit agent-PMMA binding peptide-PMMA triblock. In other uses of thisembodiment the linker 13 provides a flexible region so that the benefitagent-PMMA binding peptide-PMMA triblock can forma particularconformation or a variety of different conformations. Still, in otheruses of this embodiment the chemical and physical nature of the linker13 may be used to change the rheology of the environment surrounding thesurface 1 to which the peptide 5 is bound. Non-limiting examples oflinker domains 13 that would alter the rheology of the surroundingsurface include, hydrophobic, hydrophilic, or charged molecules.Additionally a linker domain 13 may be employed to release a benefitagent 19 from the PMMA-binding peptide 5 under various circumstances.Such circumstances may include for example, a certain range of pH, or acertain range of temperatures, or a certain range of pressures. Suchcircumstances may also include response to shock, response to thepresence of a particular molecule, especially a peptide cleavingmolecule, or the passage of time.

Referring to FIG. 2 panel A, a surface 101 is shown that could becomprised of any surface material. Non-limiting examples of suchsurfaces are metal, paper, glass and cloth. A coating 121 comprised ofin whole or in part, PMMA molecules or moieties 103 has been applied tothe surface 101 as shown. In this embodiment of the invention, aPMMA-binding peptide 105 is targeted to the coating. As in the abovedescriptions the PMMA-binding peptide 105 comprises, in whole or inpart, at least one PMMA-binding domain 107, which itself comprises, inwhole or in part, at least one PMMA-binding site 115. As describedelsewhere herein the PMMA-binding site 115 binds specifically to PMMAmolecules 103. In this embodiment the PMMA-binding site 115 binds toexposed portions of PMMA 103 in a PMMA coating 121 on attached to thesurface 101. In this embodiment the PMMA-binding peptide 105 is usefulto provide an additional coating to PMMA coating 103 already applied tothe surface 101. Non-limiting examples of the uses for this embodimentinclude a sacrificial layer to protect the PMMA coating or in the caseof multiple PMMA domains 107 and/or binding sites 115 to act as anadhesive between the PMMA coat 121 and other PMMA moieties 103 orsurfaces 101.

Similar to Panel A, FIG. 2 Panel B depicts a PMMA-binding peptide 105coupled to a PMMA coating 121 on a surface 101. The PMMA-binding peptide105 depicted in panel B further comprises a benefit agent 119. In thisembodiment of the invention, at least one, but not limited to one,benefit agent 119 is coupled to the PMMA-binding peptide 105 by acovalent, ionic or other interactive means. The PMMA bindingpeptide-benefit agent complex is in turn coupled to a PMMA moiety 103within the PMMA coating 121 on the surface 101. As described elsewhereherein the benefit agent 119 has some activity or functionally thatpersists while it is bound to the PMMA-binding peptide 105 and thecomplex is bound to the PMMA coating 121. The active benefit agent 119being brought to or near the coating 121 by the PMMA-binding peptide 105conveys its activity to the coating, modifies the coating or enhancesthe coating. In a similar embodiment, a plurality of different types ofPMMA-binding peptides 105 may be used in combination so that thedifferent benefit agents 119 corresponding to the different PMMA-bindingpeptides 105 may interact, or act in concert to produce a desirableresult.

Similar to Panel A, FIG. 2 Panel C depicts a PMMA-binding peptide 105bound to a PMMA coating 121 on a surface 101. The PMMA-binding peptide105 comprises all of the features of the PMMA-binding peptide 105depicted in Panel A, with the added feature that the peptide includes anactive domain 109 separate from the PMMA-binding domain. This activedomain 109, remains active as part of the PMMA-binding peptide 105, andfurther continues to be active when the PMMA-binding peptide 105 bindsto PMMA 103 within the PMMA coating 121. The active domain 109, byvirtue of being part of the diblock containing the PMMA-binding peptide105 and the PMMA coating 121, is active in the proximity of the coating121. This allows the coating 121 to exhibit the activity of the activedomain 109 contained in the PMMA-binding peptide 105 bound to the PMMAmolecules 103 contained within the coating 121. Additionally, as inpanel B, a benefit agent 119 may also be chemically attached to thefunctional domain containing PMMA-binding peptide 105 (not shown). Asstated above, an additional embodiment would be the use of a pluralityof different types of PMMA-binding peptides 105 to interact or act inconcert to produce a desirable result.

Referring to FIG. 3, another embodiment of the invention is shown inwhich the PMMA substrate is not bound to a larger surface. In thisembodiment the PMMA exists as a PMMA moiety 203 which may be suspendedin solution, such as cell growth media, air, water, oil, biologicalfluids, gels and the like. A PMMA-binding peptide 205 is depicted boundto the PMMA moiety 203. The PMMA-binding'peptide 205 contains within itspeptide structure at least one, but not limited to one PMMA bindingdomain 207. The PMMA binding domain 207 contains within its structure aPMMA binding site 215. PMMA binding domains 207 and PMMA binding sites215 bind PMMA moieties 203 specifically as described elsewhere herein.Binding of the PMMA-binding peptide 205 to the PMMA 203 occurs at thePMMA binding site 215 with the PMMA binding domain 207. The binding ofPMMA moieties 203 to the PMMA-binding peptide 205 forms a diblockstructure.

Other embodiments of the invention add additional elements to thedi-block formation of PMMA moiety 203 and PMMA-binding peptide 205. Onesuch embodiment, involves binding a benefit agent 219 to thePMMA-binding peptide 205. The addition of a benefit agent 219 to thediblock structure forms a triblock structure. The benefit agent 219 maybe coupled to the PMMA-binding peptide 205 by any known means, asdescribed above. The function of benefit agents 219 is discussed ingreater detail elsewhere herein.

Alternatively the benefit agent 223 may be attached to the PMMA moiety203. In this format, the benefit agent 223 is attached to the PMMAmoiety or bead 203 typically by chemical means or bonds 225. The bondmay be part of the benefit agent 223 or may be an independent structurethat is bound to the PMMA 203 for the purpose of binding the benefitagent 223. In the alternative, the bond structure 225 may be bound tothe benefit agent 223 for the purpose of binding it to the PMMA 203. Thebinding structure 225 may be a permanent bond, but in some forms of theembodiment may be easily broken under certain conditions. In other formsof the embodiment, the bond 225 may allow the benefit agent 223 to beleached from the PMMA moiety or bead 203 under certain conditions. Instill other forms of the embodiment, the bond 225 may allow the benefitagent 223 to be released over time at regular or specific timeintervals. Alternatively, the bond 225 itself may be in whole or in partbe composed of PMMA. In this way, the PMMA moiety or bead 203 may be inwhole or in part the binding structure 225. The benefit agent 223 may bepartially or fully embedded with the PMMA moiety or bead 203.

In another embodiment described by FIG. 3, the PMMA-binding peptide 205is bound the PMMA 203 as described above and the complex may optionallybe bound to a benefit agent 219 and/or 223 by any method describedelsewhere herein. The additional feature of this embodiment is at leastone or a plurality of additional active peptide domains 209 within thePMMA-binding peptide 205. Any known peptide active domain 209 can beused in this embodiment. Alternatively, the active domain 209 may be alinker domain or may function as a target domain and bind a target 217.

Additional embodiments of the invention are illustrated in FIGS. 4 and5. Referring to FIGS. 4 and 5, a plurality of PMMA-binding peptides 305may be employed to bring substances together. FIG. 4 depicts aPMMA-binding peptide 305 used to bring a PMMA containing surface 301together with another surface 333. The PMMA containing surface 301 iscomprised in whole or in part of PMMA moieties 303 At least some PMMAmoieties 303 are exposed in part or in full on the at least one side ofthe surface 301. Likewise, the non-PMMA surface or complementary surface333, is comprised in whole or in part of a known moiety 335 for whichthere exists a peptide binding-domain 329 or for which a peptide bindingdomain 329 can be designed using the methods described elsewhere herein.The amino acid structure of the PMMA-binding peptides 305 comprises inwhole or in part of at least one PMMA binding domain 307, but possiblymore than one. The PMMA binding domain 307 itself comprises at least oneor a plurality of PMMA binding sites 315. PMMA binding sites 315 areable to bind to the exposed PMMA 303 of the PMMA containing surface 301and in such way to adhere the PMMA-binding peptides 305 to surface 301.In addition to comprising one or more PMMA binding domains 307, thePMMA-binding peptide 305 of this embodiment also comprises at least one,but possibly more, target binding domains 311. The target binding domain311 specifically binds another target domain 337 in a handshake fashionallowing the complex to serve as an adhesive binding the PMMA andnon-PMMA containing surfaces together. The target binding domain 311 insome uses may be capable of binding to itself. In that case, the targetbinding domain 311 and the target domain 337 could be identical.

The complementary surface 333 is composed a known surface-exposed moietyor a complementary moiety 335, for which there is a known peptidebinding domain 329, a complementary peptide binding domain 329. Acomplementary moiety binding peptide 327 is composed of at least one,but possibly more than one, complementary moiety binding domain 329,which itself is composed of at least one but possibly more than onecomplementary moiety binding site 331. The complementary moiety bindingsite 331 binds specifically to complementary moieties 335 exposed on thecomplementary surface 333. The complementary moiety binding peptide 327is bound to the complementary surface 333 because it is composed of atleast one complementary moiety binding domain 329 which contains atleast one complementary moiety binding site 331. In addition to thecomplementary moiety binding domain 329, the complementary moietybinding peptide 327 also contains at least one but possibly more thanone target domain 337. As discussed above, the target binding domain 311of the PMMA-binding peptide 305 binds to the target domain 337.

It should be clear to one skilled in the art that the complementarysurface 333 may be composed of PMMA itself and the complementary moietybinding domain 327 could be a PMMA binding domain. This embodiment isuseful because it provides an adhesive that is specific and functionaleven in adverse circumstances among such circumstances, as not limitingexamples, are the presence of water, oil, or dirt.

FIG. 5 depicts another embodiment of the invention useful for bindingtwo surfaces together. In this embodiment neither surface needs tonecessarily contain PMMA, although that possibility is not excluded. Theprimary structure of the peptide based adhesive is similar to that shownin FIG. 4. Two surfaces are provided 433, 439. Each surface comprising atarget molecule 435, 441 either of which may or may not be the same andmay or not be PMMA. A peptide diblock is provided comprising in eachcase a target binding peptide 405 with a target binding domain 411comprising a target binding site 431. The target binding peptide 405comprises a PMMA binding domain 407 having a PMMA binding site 415,useful for binding PMMA moieties. Juxtaposing of the two surfaces in thepresence of PMMA moieties 403 results in adhesion of the surfaces thoughthe PMMA.

It will be apparent to the skilled person that this embodiment may alsobe practiced with the addition of a benefit agent(s) and/or peptidedomain(s) as describe above. This embodiment is useful because itprovides an adhesive that is specific and functional even in adversecircumstances among such circumstances, as not limiting examples, arethe presence of water, oil, or dirt.

FIG. 6 depicts an embodiment of the invention in which a surface 533 maybe coated with PMMA 503 using PMMA-binding peptide 505 containing atarget binding domain 511. FIG. 6 panel A depicts a surface 533 coatedwith a target peptide 527 that contains in part or whole a target domain537. The target peptide 527 may be applied to the surface 533 by anymethod either described herein or known in the art; one method will bedescribed in detail later when discussing FIG. 6 panel D. ThePMMA-binding peptides 505 used in this embodiment each contain, asdescribed above, at least one PMMA binding domain 507 which in turncontains at least one PMMA binding site 515. The PMMA binding site 515binds PMMA 503 specifically as described elsewhere herein. In additionto the PMMA binding domain 507, the PMMA-binding peptides 505 also eachcontain at least one but possibly more than one target binding domain511. The target binding domain used is selected, or created, usingmethods described or known, to bind specifically to the target domain537 of the target peptide 527 on the surface 533. If the PMMA-bindingpeptide 505 and PMMA moieties 503 as described are allowed to movefreely in a medium around the exposed surface 533, PMMA-binding peptide505 will adhere to the peptides 527 on the surface 533 through thebonding of the target binding domain 511 of the PMMA-binding peptide 505to the target domain 537 of the surface peptide 527. PMMA moieties inthe media will bind to the PMMA binding site 515 of the PMMA bindingdomain 507 of the PMMA-binding peptide 505 forming a diblock structure.With PMMA 503 bound to the PMMA-binding peptide 505 and it in turn boundto the suface peptides 527 that are bound to the surface 533, PMMA 503moieties will coat the surface 533.

FIG. 6 panel B, depicts the same interactions of PMMA-binding peptide505, PMMA 503 and a peptide coated surface 533, as described in panel A,with the addition of a benefit agent 517 coupled to the PMMA-bindingpeptide 505 which itself contains at least one target binding domain511. Using methods described herein this embodiment couples a benefitagent 517 to the PMMA-binding peptide 505. When the complex of thebenefit agent 517 and the PMMA-binding peptide 505 bind a PMMA moiety503 a triblock is formed. The triblock structure does not prevent thebenefit agent 517 from being functionally active or from the targetbinding domain 511 from binding the target peptide 527. The addition ofa benefit agent 517 to the PMMA-binding peptide 505 allows the surfaceto be coated with both a benefit agent 519 and PMMA moieties 503.Non-limiting examples of benefits agents 517 that may be used with thisembodiment are dyes, colorants, antimicrobials, and stain repellingmoieties.

FIG. 6 panel C, depicts the same interactions as in panel A, andprovides the addition of a benefit agent 523 bound to PMMA. In thisembodiment, the benefit agent 523 is attached to the PMMA moiety or bead503 with a bond structure 525. The bonding structure may be part of thebenefit agent 523 or may be an independent structure that is bound tothe PMMA 503 for the purpose of binding the benefit agent 523. Or in thealternative, the bond structure 525 may be bound to the benefit agent523 for the purpose of binding it to the PMMA 503. The binding structure523 may be a permanent bond, but in some forms of the embodiment may beeasily broken under certain conditions. In other forms of the embodimentthe binding structure 525 may allow the benefit agent 523 to be leachedfrom the PMMA 503 under certain conditions. In still other forms of theembodiment the binding structure might allow the benefit agent 523 to bereleased over time at regular or specific time intervals. In analternative form of this embodiment the binding structure 525 itself maybe in whole or in part be composed of PMMA. In this form, the PMMA 503may be in whole or in part the binding structure 525. The benefit agent523 may be partially or fully embedded with the PMMA 503. A triblockstructure is formed when the benefit agent 523 coupled to the PMMAmoiety 503 that is inturn bound to the PMMA-binding peptide 505. Thetriblock structure is capable of binding the target peptide as describedabove.

FIG. 6 panel D, Depicts a PMMA-binding peptide 505 and PMMA moiety orbead 503 similar to the PMMA-binding peptide 505 described in panel A.In this embodiment, the target peptide 527 depicted is not attacheddirectly to the surface 533. The target peptide 527 contains a targetbinding domain 537 as in panels A, B, and C and additionally contains asurface moiety binding domain 529. The surface moiety binding domain 529is selected to bind specifically to a known moiety that is known to beexposed on the surface 533. The surface binding moiety domain 529contains at least one, but possibly more than one, surface moiety bindsite 531. The surface moiety binding site 531 is the point of attachmentbetween the surface moiety 535 and the surface moiety binding domain529. Through the interaction of the surface moiety binding domain 529and the surface moiety 535 the PMMA-binding peptide 505 is attached tothe surface 533. Further through the binding interaction of the PMMAmoieties or beads 503 and PMMA-binding peptide 505 bound to the surface503, the surface 503 is coated with PMMA moieties or beads 503.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

The meaning of abbreviations used is as follows: “min” means minute(s),“sec” means second(s), “h” means hour(s), “μL” means microliter(s), “mL”means milliliter(s), “L” means liter(s), “nm” means nanometer(s), “mm”means millimeter(s), “cm” means centimeter(s), “μm” means micrometer(s),“mM” means millimolar, “M” means molar, “mmol” means millimole(s),“μmole” means micromole(s), “g” means gram(s), “μg” means microgram(s),“mg” means milligram(s), “g” means the gravitation constant, “rpm” meansrevolutions per minute, “pfu” means plague forming unit, “BSA” meansbovine serum albumin, “ELISA” means enzyme linked immunosorbent assay,“IPTG” means isopropyl β-D-thiogalactopyranoside, “A” means absorbance,“A₄₅₀” means the absorbance measured at a wavelength of 450 nm, “TBS”means Tris-buffered saline, “TBST-X” means Tris-buffered salinecontaining Tween® 20 where “X” is the weight percent of Tween® 20,“Xgal” means 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, “SEM”means standard error of the mean, “vol %” means volume percent.

General Methods:

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, byT. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984,and by Ausubel, F. M. et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are also well known in the art. Techniques suitablefor use in the following Examples may be found in Manual of Methods forGeneral Bacteriology, Phillipp Gerhardt, R. G. E. Murray, Ralph N.Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. BriggsPhillips, eds., American Society for Microbiology, Washington, D.C.,1994, or by Thomas D. Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition, Sinauer Associates, Inc., Sunderland,Mass., 1989.

All reagents, restriction enzymes and materials used for the growth andmaintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), LifeTechnologies (Rockville, Md.), or Sigma Chemical Company (St. Louis,Mo.), unless otherwise specified.

Example 1 Selection of Polymethylmethacrylate (PMMA)-Binding PeptidesUsing Biopanning

The purpose of this Example was to identify phage peptides that bind topolymethylmethacrylate (PMMA) using a modified phage display biopanningmethod.

Phage Display Peptide Libraries:

The phage libraries used in the present invention, Ph.D.-12™ PhageDisplay Peptide Library Kit and Ph.D.-7™ Phage Display Library Kit, werepurchased from New England BioLabs (Beverly, Mass.). These kits arebased on a combinatorial library of random peptide 7 or 12-mers fused toa minor coat protein (pIII) of M13 phage. The displayed peptide isexpressed at the N-terminus of pill, such that after the signal peptideis cleaved, the first residue of the coat protein is the first residueof the displayed peptide. The Ph.D.-7 and Ph.D.-12 libraries consist ofapproximately 2.8×10⁹ and 2.7×10⁹ sequences, respectively. A volume of10 μL contains about 55 copies of each peptide sequence. Each initialround of experiments was carried out using the original library providedby the manufacturer in order to avoid introducing any bias into theresults.

Biopanning Against a PMMA Surface:

The PMMA materials used were ⅛ inch (32 mm) thick, ½ inch (12.7 mm)diameter disks of Lucite® sheet (obtained from E.I. du Pont de Nemoursand Co., Wilmington, Del.) and a dot blot apparatus (obtained fromSchleicher & Schuell, Keene, N.H.). The following protocol was used forbiopanning against the PMMA disk. The PMMA disk was placed in a tubefilled with 5 mL of 90% isopropanol for 30 min at room temperature andthen washed 5 times for 10 min each with deionized water. Then, 5 mL ofblocking buffer consisting of 1 mg/mL BSA in TBST containing 0.5% Tween®20 (TBST-0.5%) was added to the tube and incubated for 1 h at 4° C.

The disk was washed 5 times with TBST-0.5% and then 2 mL of TBST-0.5%containing 1 mg/mL BSA was added to each well. Then, 10 μL of theoriginal phage library (2×10¹¹ pfu), either the 12-mer or 7-mer library,was added to the disk and incubated for 15 min at room temperature. Thedisk was washed 10 times with TBST-0.5%. The disk was then transferredto a clean tube, 2 mL of a non-specific elution buffer consisting of 1mg/mL BSA in 0.2 M glycine-HCl, pH 2.2, was added to the tube andincubated for 10 min. The disk was washed three more times with theelution buffer and then washed three times with TBST-0.5%. The disk,which had acid resistant phage peptides still attached, was used todirectly infect the host cells E. coli ER 2738 (New England BioLabs,Beverly, Mass.), for phage amplifications. The disk was incubated withan overnight E. coli ER2738 culture diluted 1:100 in LB medium, at 37°C. for 4.5 h. After this time, the cell culture was centrifuged for 30 sand the upper 80% of the supernatant was transferred to a fresh tube, ⅙volume of PEG/NaCl (20% polyethylene glycol-800, obtained from SigmaChemical Co. St. Louis, Mo., 2.5 M sodium chloride) was added, and thephage was allowed to precipitate overnight at 4° C. The precipitate wascollected by centrifugation at 10,000×g at 4° C. and the resultingpellet was resuspended in 1 mL of TBS. This was the first round ofamplified stock. The amplified first round phage stock was then titeredaccording to the method described below. For the next round ofbiopanning, more than 2×10¹¹ pfu of phage stock from the first round wasused. The biopanning process was repeated for 3 to 4 rounds depending onthe experiments.

After the acid wash steps in the final round of biopanning, the PMMAdisk was used to directly infect 500 μL of mid-log phase bacterial hostcells, E. coli ER2738, which were then grown in LB medium for 20 min andthen mixed with 3 mL of agarose top (LB medium with 5 mM MgCl₂, and 0.7%agarose) at 45° C. This mixture was spread onto a LB medium/IPTG/S-Gal™plate (LB medium with 15 g/L agar, 0.05 g/L IPTG, and 0.04 g/L S-Gal™)and incubated overnight at 37° C. The black plaques were counted tocalculate the phage titer. The single black plaques were randomly pickedfor DNA isolation and sequencing analysis. The amino acid sequences ofthese high affinity, PMMA-binding phage peptides are given in Table 7.

TABLE 7 Amino Acid Seauences of High Affinity PMMA-Binding Phage Peptides from the 7- and 12-Mer Libraries Clone IDAmino Acid Sequence SEQ ID NO: A09 IPWWNIRAPLNA 1 D09 TAVMNVVNNQLS 2 A03VPWWAPSKLSMQ 3 A06 MVMAPHTPRARS 4 B04 TYPNWAHLLSHY 5 B09 TPWWRIT 6 B01DLTLPFH 7 PB411 GTSIPAM 8 P307 HHKHVVA 9 P410 HHHKHFM 10 P202 HHHRHQG 11PNM407 HHWHAPR 12

Example 2 Characterization of PMMA-Binding Phage Peptide Clones by ELISA

Enzyme-linked immunosorbent assay (ELISA) was used to evaluate thePMMA-binding affinity of the selected phage-peptide clones identified inExample 1 along with a skin-1 phage clone TPFHSPENAPGS (given as SEQ IDNO:81), which served as a control.

An empty 96-well apparatus, a Minifold I Dot-Blot System from Schleicher& Schuell, Inc. (Keene, N.H.) was used as the PMMA surface. For eachclone to be tested, the well was incubated for 1 h at room temperaturewith 200 μL of blocking buffer, consisting of 2% non-fat dry milk inTBS. The blocking buffer was removed by inverting the systems andblotting them dry with paper towels. The wells were rinsed 6 times withwash buffer consisting of TBST-0.5%. The wells were filled with 200 μLof TBST-0.5% containing 1 mg/mL BSA and then 10 μL (over 10¹² copies) ofpurified phage stock was added to each well. The samples were incubatedat 37° C. for 15 min with slow shaking. The non-binding phage wasremoved by washing the wells 10 to 20 times with TBST-0.5%. Then, 100 μLof horseradish peroxidase/anti-M13 antibody conjugate (Amersham USA,Piscataway, N.J.), diluted 1:500 in the blocking buffer, was added toeach well and incubated for 1 h at room temperature. The conjugatesolution was removed and the wells were washed 6 times with TBST-0.05%.TMB substrate (200 μL), obtained from Pierce Biotechnology (Rockford,Ill.) was added to each well and the color was allowed to develop forbetween 5 to 30 min, typically for 10 min, at room temperature. Then,stop solution (200 μL of 2 M H₂SO₄) was added to each well and thesolution was transferred to a 96-well plate and the A₄₅₀ was measuredusing a microplate spectrophotometer (Molecular Devices, Sunnyvale,Calif.). The resulting absorbance values, reported as the mean of atleast three replicates, and the standard error of the mean (SEM) aregiven in Table 8.

TABLE 8 Results of ELISA Assay SEQ ID PMMA Clone ID NO: A₄₅₀ SEM Skin-181 0.127 0.057 (Control) A09 1 2.227 0.020 D09 2 2.037 0.057 A03 3 0.7620.081 A06 4 2.09 0.115 B04 5 2.095 0.065 B09 6 2.261 0.016 B01 7 2.1120.060

The results demonstrate that all of the PMMA-binding phage peptidestested had a significantly higher binding affinity for PMMA than thecontrol skin-1 peptide.

Example 3

Determination of the PMMA-Binding Affinity of PMMA-Binding Peptides

The purpose of this Example was to determine the affinity of thePMMA-binding peptides for PMMA surfaces, measured as MB₅₀ values, usingan ELISA assay. The term “MB₅₀” refers to the concentration of thebinding peptide that gives a signal that is 50% of the maximum signalobtained in an ELISA-based binding assay. The MB₅₀ provides anindication of the strength of the binding interaction or affinity of thecomponents of the complex. The lower the value of MB₅₀, the stronger theinteraction of the peptide with the PMMA substrate.

PMMA-binding peptide A09 (SEQ ID NO:1) was synthesized by Synpep Inc.(Dublin, Calif.). The peptide was biotinylated by adding a biotinylatedlysine residue at the C-terminus of the amino acid binding sequence fordetection purposes and an amidated cysteine was added to the C-terminusof the sequence.

MB₅₀ Measurement of PMMA-Binding Peptide A09:

The MB₅₀ measurements of biotinylated peptide binding to PMMA were doneusing the 96-well apparatus described in Example 2. The 96-wells wereblocked with blocking buffer (SuperBlock™ from Pierce Chemical Co.,Rockford, Ill.) at room temperature for 1 h, followed by six washes withTBST-0.5%, 2 min each, at room temperature. Various concentrations ofbiotinylated, binding peptide were added to each well, incubated for 15min at 37° C., and washed six times with TBST-0.5%, 2 min each, at roomtemperature. Then, streptavidin-horseradish peroxidase (HRP) conjugate(Pierce Chemical Co., Rockford, Ill.) was added to each well (1.0 μg perwell), and incubated for 1 h at room temperature. After the incubation,the wells were washed six times with TBST-0.5%, 2 min each at roomtemperature. Finally, the color development and the absorbancemeasurements were performed as described in Example 2.

The results were plotted as A₄₅₀ versus the concentration of peptideusing GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego, Calif.).The MB₅₀ values were calculated from Scatchard plots and are shown Table9.

MB₅₀ Measurement of Tetramer PMMA-Binding Peptide A09:

For the MB₅₀ measurement of the peptide A09 tetramer, the PMMA surfaceand all the binding conditions were the same as described above. Thetetrameric-A09 peptide complex was prepared by mixing streptavidin-HRPand biotinylated peptide A09 in a 1:4 molar ratio. After all theblocking and washing steps, various concentrations ofStreptavidin/(A09)₄ complex were added to each well, incubated for 15min at 37° C., and washed six times with TBST-0.5%, 2 min each, at roomtemperature. Then, color development and the absorbance measurementswere performed as described in Example 2. The results were plotted asA₄₅₀ versus the concentration of peptide complex using GraphPad Prism4.0 (GraphPad Software, Inc., San Diego, Calif.). The MB₅₀ values werecalculated from Scatchard plots and are shown in Table 9.

TABLE 9 Summary of MB₅₀ Values for PMMA-Binding Peptides Binding PeptideSubstrate MB_(50,) M A09 PMMA 5.9 × 10⁻⁸ Streptavidin/(A09)₄ PMMA 3.9 ×10⁻⁹

The results demonstrate that the binding affinity of the PMMA-bindingpeptide, A09, and the straptavidin/A09 tetramer complex for PMMA washigh. The use of multiple copies of the binding peptide in the A09tetramer complex increased the binding affinity by more than 10-fold.

1. A method for delivering a functional peptide to a PMMA (“polymethylmethacrylate”) surface comprising, a) providing a peptide reagent havinga general structure selected from the group consisting of:PMMA_(m)-(PmBP)_(n);  A)PMMA_(m)-(PmBP-BAp)_(n);  B)PMMA_(m)-(PmBP-AD)_(n);  C)PMMA_(m)-(PmBP-TBD)_(n);  D)PMMA_(m)-(PmBP-L-BA)_(n); and  E)PMMA_(m)-[(PmBP)_(q)-(L)_(x)-(PmBP)_(r)]_(n)-L-BA;  F) wherein: i) PMMAis a polymethylmethacrylate moiety ii) PmBP is a PMMA binding peptidehaving a PMMA binding domain; iii) BA is at least one benefit agent; iv)AD is at least one active domain incorporated into a PMMA bindingpeptide; v) TBD is at least one target binding domain incorporated intoa PMMA binding peptide; vi) L is a linker molecule; vii) m=the number ofPMMA moieties available for binding; viii) n=is less than m; xi) p=1-20;x) x=1-20; and xi) r=1-50; b) providing a PMMA_(m) surface; and c)contacting the peptide reagent of (a) with the PMMA_(m) surface of (b)under conditions whereby PmBP binds to the PMMA_(m) surface.
 2. A methodfor delivering a benefit agent to a PMMA_(m) substrate comprising: a)providing a peptide reagent comprising a PMMA_(m)-binding peptide(“PmBP”) having the general structure selected from the group consistingof:PMMA_(m)-(PmBP-BAp)_(n);  A)PMMA_(m)-(PmBP-L-BA)_(n); and  B)PMMA_(m)-[(PmBP)_(q)-(L)_(x)-(PmBP)_(r)]_(n)-L-BA;  C) wherein: i) PMMAis a polymethacrylate moiety ii) PmBP is a PMMA binding peptide having aPMMA binding domain; iii) BA is at least one benefit agent; vi) L is alinker molecule; vii) m=the number of PMMA moieties available forbinding; viii) n=is less than or equal to m; xi) p=1-20; x) x=1-20; andxi) r=1-50; b) providing a PMMA_(m) substrate; and c) contacting thepeptide reagent of (a) with the substrate of (b) under conditionswhereby the PmBP binds to the PMMA substrate.
 3. The method according toclaim 2 wherein the at least one benefit agent is selected from thegroup consisting of pharmaceuticals, markers, colorants, conditionersand fragrances.
 4. A method for preparing a PMMA surface coated with oneor more peptide reagents comprising: a) providing a peptide reagentaccording to a general structure selected from the group consisting of:PMMA_(m)-(PmBP)_(n);  A)PMMA_(m)-(PmBP-BAp)_(n);  B)PMMA_(m)-(PmBP-AD)_(n);  C)PMMA_(m)-(PmBP-TBD)_(n);  D)PMMA_(m)-(PmBP-L-BA)_(n); and  E)PMMA_(m)-[(PmBP)_(q)-(L)_(x)-(PmBP)_(r)]_(n)-L-BA;  F) wherein: i) PMMAis a polymethylmacrylate moiety ii) PmBP is a PMMA binding peptidehaving a PMMA binding domain; iii) BA is at least one benefit agent; iv)AD is at least one active domain incorporated into a PMMA bindingpeptide; v) TBD is at least one target binding domain incorporated intoa PMMA binding peptide; vi) L is a linker molecule; vii) m=the number ofPMMA moieties available for binding; viii) n=is less than or equal to m;xi) p=1-20; x) x=1-20; xi) r=1-50; and b) contacting the peptide reagentof (a) with a PMMA surface under conditions whereby the peptide reagentbinds to the PMMA surface.